Crystalline forms of a PI3K inhibitor

ABSTRACT

The present invention is related to crystalline forms of (S)-7-(1-(9H-purin-6-ylamino) ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one which is a PI3K inhibitor useful in the treatment of cancer and other diseases.

FIELD OF THE INVENTION

The present invention is related to crystalline forms of(S)-7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-onewhich is a PI3K inhibitor useful in the treatment of cancer and otherdiseases.

BACKGROUND OF THE INVENTION

The compound(S)-7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-onehaving Formula I:

is a phosphoinositide 3-kinase (PI3K) inhibitor useful in the treatmentof various diseases including cancer. The compound of Formula I, as wellas its preparation and use, have been described in US Pat. App. Pub. No.2011/0015212, which is incorporated herein by reference in its entirety.For the development of a drug, it is typically advantageous to employ aform of the drug having desirable properties with respect to itspreparation, purification, reproducibility, stability, bioavailability,and other characteristics. Accordingly, the crystalline forms of thecompound of Formula I provided herein help satisfy the ongoing need forthe development of PI3K inhibitors for the treatment of seriousdiseases.

SUMMARY OF THE INVENTION

The present invention provides a crystalline form of the compound ofFormula I:

which is any one of Forms I, II, III, IV, V, VI, VII, VIII, IX, X, XI,XII, and XIII described herein.

The present invention further provides a crystalline form of thecompound of Formula I which is hydrated.

The present invention further provides a crystalline form of thecompound of Formula I which is a hemihydrate.

The present invention further provides a composition comprising acrystalline form of the invention and at least one pharmaceuticallyacceptable carrier.

The present invention further provides a process for preparing acrystalline form of the invention.

The present invention further provides a method of treating a diseaseassociated with abnormal expression or activity of a PI3K kinase in apatient, comprising administering to the patient a therapeuticallyeffective amount of a crystalline form of the invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an XRPD pattern for Form I.

FIG. 2 shows the results of a DSC experiment for Form I.

FIG. 3 shows the results of a TGA experiment for Form I.

FIG. 4 shows the results of a DVS experiment for Form I; first cycle isshown in the upper panel and second cycle is shown in the lower panel.

FIG. 5 shows an XRPD pattern for Form II.

FIG. 6 shows the results of a TGA experiment for Form II.

FIG. 7 shows an XRPD pattern for Form III.

FIG. 8 shows the results of a DSC experiment for Form III; first cycleis shown in the upper panel and second cycle is shown in the lowerpanel.

FIG. 9 shows the results of a TGA experiment for Form III.

FIG. 10 shows an XRPD pattern for Form IV.

FIG. 11 shows the results of a DSC experiment for Form IV; first cycleis shown in the upper panel and second cycle is shown in the lowerpanel.

FIG. 12 shows the results of a TGA experiment for Form IV.

FIG. 13 shows an XRPD pattern for Form V.

FIG. 14 shows the results of a DSC experiment for Form V; first cycle isshown in the upper panel and second cycle is shown in the lower panel.

FIG. 15 shows the results of a TGA experiment for Form V.

FIG. 16 shows an XRPD pattern for Form VI.

FIG. 17 shows the results of a DSC experiment for Form VI; first cycleis shown in the upper panel and second cycle is shown in the lowerpanel.

FIG. 18 shows the results of a TGA experiment for Form VI.

FIG. 19 shows an XRPD pattern for Form VII.

FIG. 20 shows the results of a TGA experiment for Form VII.

FIG. 21 shows the results of a DSC experiment for Form VII; first cycleis shown in the upper panel and second cycle is shown in the lowerpanel.

FIG. 22 shows an XRPD pattern for Form VIII.

FIG. 23 shows the results of a DSC experiment for Form VIII; first cycleis shown in the upper panel and second cycle is shown in the lowerpanel.

FIG. 24 shows the results of a TGA experiment for Form VIII.

FIG. 25 shows an XRPD pattern for Form IX.

FIG. 26 shows the results of a DSC experiment for Form IX.

FIG. 27 shows the results of a TGA experiment for Form IX.

FIG. 28 shows an XRPD pattern for Form X.

FIG. 29 shows the results of a DSC experiment for Form X.

FIG. 30 shows the results of a TGA experiment for Form X.

FIG. 31 shows an XRPD pattern for Form XI.

FIG. 32 shows the results of a DSC experiment for Form XI; first cycleis shown in the upper panel and second cycle is shown in the lowerpanel.

FIG. 33 shows an XRPD pattern for Form XII.

FIG. 34 shows the results of a DSC experiment for Form XII.

FIG. 35 shows an XRPD pattern for Form XIII.

FIG. 36 shows the results of a DSC experiment for Form XIII.

DETAILED DESCRIPTION

The present invention relates to, inter alia, crystalline forms of thePI3K inhibitor(S)-7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-onehaving Formula I:

which are useful, for example, in the preparation of solid dosage formsof the above compound for the treatment of various diseases, includingcancer.

Typically, different crystalline forms of the same substance havedifferent bulk properties relating to, for example, hygroscopicity,solubility, stability, and the like. Forms with high melting pointsoften have good thermodynamic stability which is advantageous inprolonging shelf-life drug formulations containing the solid form. Formswith lower melting points often are less thermodynamically stable, butare advantageous in that they have increased water solubility,translating to increased drug bioavailability. Forms that are weaklyhygroscopic are desirable for their stability to heat and humidity andare resistant to degradation during long storage. Anhydrous forms areoften desirable because they can be consistently made without concernfor variation in weight or composition due to varying solvent or watercontent. On the other hand, hydrated or solvated forms can beadvantageous in that they are less likely to be hygroscopic and may showimproved stability to humidity under storage conditions.

As used herein, “crystalline form” is meant to refer to a certainlattice configuration of a crystalline substance. Different crystallineforms of the same substance typically have different crystallinelattices (e.g., unit cells) which are attributed to different physicalproperties that are characteristic of each of the crystalline forms. Insome instances, different lattice configurations have different water orsolvent content. The different crystalline lattices can be identified bysolid state characterization methods such as by X-ray powder diffraction(XRPD). Other characterization methods such as differential scanningcalorimetry (DSC), thermogravimetric analysis (TGA), dynamic vaporsorption (DVS), solid state NMR, and the like further help identify thecrystalline form as well as help determine stability and solvent/watercontent.

Crystalline forms of a substance include both solvated (e.g., hydrated)and non-solvated (e.g., anhydrous) forms. A hydrated form is acrystalline form that includes water in the crystalline lattice.Hydrated forms can be stoichiometric hydrates, where the water ispresent in the lattice in a certain water/molecule ratio such as forhemihydrates, monohydrates, dihydrates, etc. Hydrated forms can also benon-stoichiometric, where the water content is variable and dependent onexternal conditions such as humidity.

Crystalline forms are most commonly characterized by XRPD. An XRPDpattern of reflections (peaks) is typically considered a fingerprint ofa particular crystalline form. It is well known that the relativeintensities of the XRPD peaks can widely vary depending on, inter alia,the sample preparation technique, crystal size distribution, filters,the sample mounting procedure, and the particular instrument employed.In some instances, new peaks may be observed or existing peaks maydisappear, depending on the type of instrument or the settings (forexample, whether a Ni filter is used or not). As used herein, the term“peak” refers to a reflection having a relative height/intensity of atleast about 4% of the maximum peak height/intensity. Moreover,instrument variation and other factors can affect the 2-theta values.Thus, peak assignments, such as those reported herein, can vary by plusor minus about 0.2° (2-theta), and the term “substantially” as used inthe context of XRPD herein is meant to encompass the above-mentionedvariations.

In the same way, temperature readings in connection with DSC, TGA, orother thermal experiments can vary about ±4° C. depending on theinstrument, particular settings, sample preparation, etc. For example,with DSC it is known that the temperatures observed will depend on therate of the temperature change as well as the sample preparationtechnique and the particular instrument employed. Thus, the valuesreported herein related to DSC thermograms can vary, as indicated above,by ±4° C. Accordingly, a crystalline form reported herein having a DSCthermogram “substantially” as shown in any of the Figures is understoodto accommodate such variation.

The compound of Formula I can be isolated in numerous crystalline forms,including crystalline forms which are anhydrous, hydrated, non-solvated,or solvated. Example hydrates include hemihydrates, monohydrates,dihydrates, and the like. In some embodiments, the crystalline forms ofthe compound of Formula I are anhydrous and non-solvated. By “anhydrous”is meant that the crystalline form of the compound of Formula I containsessentially no bound water in the crystal lattice structure, i.e., thecompound does not form a crystalline hydrate.

In some embodiments, the crystalline forms of the invention aresubstantially isolated. By “substantially isolated” is meant that aparticular crystalline form of the compound of Formula I is at leastpartially isolated from impurities. For example, in some embodiments acrystalline form of the invention comprises less than about 50%, lessthan about 40%, less than about 30%, less than about 20%, less thanabout 15%, less than about 10%, less than about 5%, less than about2.5%, less than about 1%, or less than about 0.5% of impurities.Impurities generally include anything that is not the substantiallyisolated crystalline form including, for example, other crystallineforms and other substances.

In some embodiments, a crystalline form of the compound of Formula I issubstantially free of other crystalline forms. The phrase “substantiallyfree of other crystalline forms” means that a particular crystallineform of the compound of Formula I comprises greater than about 80%,greater than about 90%, greater than about 95%, greater than about 98%,greater than about 99% or greater than about 99.5% by weight of theparticular crystalline form.

Crystalline Form I

In some embodiments, the crystalline form of the compound of Formula Iis Form I. This crystalline form can be generally prepared as describedin Example 1.

Crystalline Form I can be identified by unique signatures with respectto, for example, X-ray powder diffraction (XRPD), differential scanningcalorimetry (DSC), thermogravimetric analysis (TGA), and dynamic vaporsorption (DVS). In some embodiments, crystalline Form I is characterizedby an XRPD pattern substantially as shown in FIG. 1. Peaks from the XRPDpattern are listed in Table 1.

In some embodiments, crystalline Form I is characterized by an XRPDpattern comprising a peak, in terms of 2θ, at 10.0°±0.2°. In someembodiments, crystalline Form I has an XRPD pattern comprising thefollowing peaks, in terms of 2θ: 10.0°±0.2°; 12.6°±0.2°; 15.6°±0.2°; and18.0°±0.2°. In some embodiments, crystalline Form I has an XRPD patterncomprising 2 or more, 3 or more, or 4 or more of the following peaks, interms of 2θ: 10.0°±0.2°; 11.7°±0.2°; 12.6°±0.2°; 15.1°±0.2°; 15.6°±0.2°;18.0°±0.2°; 21.2°±0.2°; 22.6°±0.2°; 24.0°±0.2°; and 28.0°±0.2°.

In some embodiments, Form I is characterized by a DSC thermogramcomprising an endothermic peak having a maximum at about 183° C. In someembodiments, crystalline Form I has a DSC thermogram substantially asshown in FIG. 2.

In some embodiments, crystalline Form I has a TGA trace substantially asshown in FIG. 3.

In some embodiments, crystalline Form I has a DVS trace substantially asshown in FIG. 4.

Crystalline Form II

In some embodiments, the crystalline form of the compound of Formula Iis Form II. Crystalline Form II can be prepared by combining Form I withan alcohol such as isopropryl alcohol and optionally heating theresulting mixture.

Crystalline Form II can be identified by unique signatures with respectto, for example, XRPD, DSC, and TGA. In some embodiments, crystallineForm II is characterized by an XRPD pattern substantially as shown inFIG. 5. Peaks from the XRPD pattern are listed in Table 11.

In some embodiments, crystalline Form II is characterized by an XRPDpattern comprising a peak, in terms of 2θ, at 9.2°±0.2°. In someembodiments, crystalline Form II has an XRPD pattern comprising thefollowing peaks, in terms of 2θ: 14.8°±0.2°; 18.5°±0.2°; 19.3°±0.2°; and22.8°±0.2°. In some embodiments, crystalline Form II has an XRPD patterncomprising 2 or more, 3 or more, or 4 or more of the following peaks, interms of 2θ: 9.2°±0.2°; 11.1°±0.2°; 14.8°±0.2°; 15.8°±0.2°; 19.3°±0.2°;20.8°±0.2°; 21.7°±0.2°; 22.8°±0.2°; and 25.6°±0.2°.

In some embodiments, crystalline Form II of the compound of Formula Ihas a TGA trace substantially as shown in FIG. 6.

Crystalline Form III

In some embodiments, the crystalline form of the compound of Formula Iis Form III. Crystalline Form III can be prepared by combining Form Iwith isopropyl acetate. The resulting mixture can be optionally heated.

Crystalline Form III can be identified by unique signatures with respectto, for example, XRPD, DSC, and TGA. For example, crystalline Form IIIis characterized by an XRPD pattern substantially as shown in FIG. 7.Peaks from the XRPD pattern are listed in Table 12.

In some embodiments, crystalline Form III is characterized by an XRPDpattern comprising a peak, in terms of 2θ, at 10.9°±0.2°. In someembodiments, crystalline Form III has an XRPD pattern comprising thefollowing peaks, in terms of 2θ: 10.9°±0.2°; and 21.8°±0.2°. In someembodiments, crystalline Form III has an XRPD pattern comprising 2 ormore, 3 or more, or 4 or more of the following peaks, in terms of 2θ:10.9°±0.2°; 11.3°±0.2°; 12.3°±0.2°; 13.9°±0.2°; 18.6°±0.2°; 21.0°±0.2°;21.8°±0.2°; 24.6°±0.2°; and 28.4°±0.2°.

In some embodiments, Form III is characterized by a DSC thermogramcomprising an endothermic peak having a maximum at about 133° C. In someembodiments, crystalline Form III has a DSC thermogram substantially asshown in FIG. 8 (upper).

In some embodiments, crystalline Form III has a TGA trace substantiallyas shown in FIG. 9.

Crystalline Form IV

In some embodiments, the crystalline form of the compound of Formula Iis Form IV. Crystalline Form IV can be prepared by combining Form I withtoluene. The resulting mixture can be optionally heated.

Crystalline Form IV of the compound of Formula I can be identified byunique signatures with respect to, for example,)(RFD, DSC, and TGA. Insome embodiments, crystalline Form IV is characterized by an XRPDpattern substantially as shown in FIG. 10.

In some embodiments, crystalline Form IV is characterized by an XRPDpattern comprising a peak, in terms of 2θ, at 8.8°±0.2°. In someembodiments, crystalline Form IV of the compound of Formula I has anXRPD pattern comprising the following peaks, in terms of 2θ: 5.9°±0.2°;8.8°±0.2°; 17.7°±0.2°; and 23.6°±0.2°. In some embodiments, crystallineForm IV of the compound of Formula I has an XRPD pattern comprising 2 ormore, 3 or more, or 4 or more of the following peaks, in terms of 2θ:5.9°±0.2°; 8.8°±0.2°; 9.1°±0.2°; 17.7°±0.2°; 23.6°±0.2°; 26.4°±0.2°;26.8°±0.2°; and 29.6°±0.2°.

In some embodiments, Form IV characterized by a DSC thermogramcomprising an endothermic peak having a maximum at about 153° C. In someembodiments, crystalline Form IV has a DSC thermogram substantially asshown in FIG. 11.

In some embodiments, crystalline Form IV has a TGA trace substantiallyas shown in FIG. 12.

Crystalline Form V

In some embodiments, the crystalline form of the compound of Formula Iis Form V. Crystalline Form V can be prepared by combining Form I withisobutyl acetate. The resulting mixture can be optionally heated.

Crystalline Form V can be identified by unique signatures with respectto, for example, XRPD, DSC, and TGA. In some embodiments, crystallineForm V is characterized by an XRPD pattern substantially as shown inFIG. 13. Peaks from the XRPD pattern are listed in Table 14.

In some embodiments, crystalline Form V is characterized by an XRPDpattern comprising a peak, in terms of 2θ, at 12.0°±0.2°. In someembodiments, crystalline Form V has an XRPD pattern comprising thefollowing peaks, in terms of 2θ: 12.0°±0.2°; 13.6°±0.2°; 17.5°±0.2°; and22.9°±0.2°. In some embodiments, crystalline Form V has an XRPD patterncomprising 2 or more, 3 or more, or 4 or more of the following peaks, interms of 2θ: 11.1°±0.2°; 12.0°±0.2°; 13.6°±0.2°; 15.4°±0.2°; 17.5°±0.2°;19.9°±0.2°; 22.4°±0.2°; 22.9°±0.2°; and 24.8°±0.2°.

In some embodiments, Form V is characterized by a DSC thermogramcomprising an endothermic peak having a maximum at about 153° C. In someembodiments, crystalline Form V of the compound of Formula I ischaracterized by the DSC thermogram substantially as shown in FIG. 14(upper).

In some embodiments, crystalline Form V has a TGA trace substantially asshown in FIG. 15.

Crystalline Form VI

In some embodiments, the crystalline form of the compound of Formula Iis crystalline Form VI. This crystalline form appears to be hydrated,e.g., a hemihydrate based on, for example, TGA data supplied herein.

In some embodiments, the invention provides a process for preparingcrystalline Form VI comprising combining crystalline Form I with water.In some embodiments, the process further comprises heating the mixtureresulting from the combining of crystalline Form I and water. In someembodiments, the mixture can be heated to between about 30 and about 70°C., between about 40 and about 60° C., or at about 50° C. to yield FormVI.

In some embodiments, crystalline Form VI can be identified by uniquesignatures with respect to, for example, XRPD, DSC, and TGA. In someembodiments, crystalline Form VI is characterized by an XRPD patternsubstantially as shown in FIG. 16. Peaks from the XRPD pattern arelisted in Table 15.

In some embodiments, crystalline Form VI is characterized by an XRPDpattern comprising a peak, in terms of 2θ, at 10.7°±0.2°. In someembodiments, crystalline Form VI has an XRPD pattern comprising thefollowing peaks, in terms of 2θ: 10.7°±0.2°; 14.6°±0.2°; 19.1°±0.2°; and23.9°±0.2°. In some embodiments, crystalline Form VI has an XRPD patterncomprising 2 or more, 3 or more, or 4 or more of the following peaks, interms of 2θ: 10.7°±0.2°; 14.6°±0.2°; 16.0°±0.2°; 19.1°±0.2°; 22.4°±0.2°;23.9°±0.2°; 24.5°±0.2°; 26.7°±0.2°; 29.1°±0.2°; 30.3°±0.2°; and34.7°±0.2°.

In some embodiments, crystalline Form VI is characterized by the DSCthermogram substantially as shown in FIG. 17 (upper).

In some embodiments, crystalline Form VI has a TGA trace substantiallyas shown in FIG. 18. In some embodiments, the Form VI has a TGA traceshowing about 2.6% weight loss up to about 100° C.

Crystalline Form VII

In some embodiments, the crystalline form of the compound of Formula Iis Form VII. Crystalline Form VII of the compound of Formula I can beprepared by combining Form I with 1,4-dioxane, methyl isobutyl ketone,or water, or mixtures of any of the aforementioned. The resultingmixture can be optionally heated.

Crystalline Form VII can be identified by unique signatures with respectto, for example, XRPD, DSC, and TGA. In some embodiments, crystallineForm VII of the compound of Formula I is characterized by an XRPDpattern substantially as shown in FIG. 19. Peaks from the XRPD patternare listed in Table 16.

In some embodiments, crystalline Form VII is characterized by an XRPDcomprising a peak, in terms of 2θ, at 12.0°±0.2°. In some embodiments,crystalline Form VII has an XRPD pattern comprising the following peaks,in terms of 2θ: 12.0°±0.2°; 15.1°±0.2°; 17.8°±0.2°; and 24.6°±0.2°. Insome embodiments, crystalline Form VII of the compound of Formula I hasan XRPD pattern comprising 2 or more, 3 or more, or 4 or more of thefollowing peaks, in terms of 2θ: 8.8°±0.2°; 11.0°±0.2°; 12.0°±0.2°;15.1°±0.2°; 15.8°±0.2°; 16.2°±0.2°; 17.8°±0.2°; 18.5°±0.2°; 19.5°±0.2°;22.1°±0.2°; 24.6°±0.2°; and 25.9°±0.2°.

In some embodiments, Form VII is characterized by a DSC thermogramcomprising an endothermic peak having a maximum at about 123° C. In someembodiments, crystalline Form VII is characterized by the DSC thermogramsubstantially as shown in FIG. 21 (upper).

In some embodiments, crystalline Form VII has a TGA trace substantiallyas shown in FIG. 20.

Crystalline Form VIII

In some embodiments, the crystalline form of the compound of Formula Iis Form VIII. Crystalline Form VIII can be prepared by combining Form Iwith n-butyl alcohol. The resulting mixture can be optionally heated.

Crystalline Form VIII can be identified by unique signatures withrespect to, for example, XRPD, DSC, and TGA. In some embodiments,crystalline Form VIII is characterized by an XRPD pattern substantiallyas shown in FIG. 22. Peaks from the XRPD pattern are listed in Table 17.

In some embodiments, crystalline Form VIII is characterized by an XRPDpattern comprising a peak, in terms of 2θ, at 10.9°±0.2°. In someembodiments, crystalline Form VIII has an XRPD pattern comprising thefollowing peaks, in terms of 2θ: 10.9°±0.2°; 11.7°±0.2°; 21.5°±0.2°; and22.6°±0.2°. In some embodiments, crystalline Form VIII has an XRPDpattern comprising 2 or more, 3 or more, or 4 or more of the followingpeaks, in terms of 2θ: 3.9°±0.2°; 8.2°±0.2°; 10.9°±0.2°; 11.7°±0.2°;14.4°±0.2°; 16.1°±0.2°; 17.5°±0.2°; 19.7°±0.2°; 21.5°±0.2°; 22.6°±0.2°;and 25° 0.3±0.2°.

In some embodiments, Form VIII is characterized by a DSC thermogramcomprising an endothermic peak having a maximum at about 176° C. In someembodiments, crystalline Form VIII characterized by the DSC thermogramsubstantially as shown in FIG. 23 (upper).

In some embodiments, crystalline Form VIII has a TGA trace substantiallyas shown in FIG. 24.

Crystalline Form IX

In some embodiments, the crystalline form of the compound of Formula Iis Form IX. Crystalline Form IX can be prepared by combining Form I withmethyl isobutyl ketone. The resulting mixture can be optionally heated.

Crystalline Form IX can be identified by unique signatures with respectto, for example, XRPD, DSC, and TGA. In some embodiments, crystallineForm IX of the compound of Formula I is characterized by an XRPD patternsubstantially as shown in FIG. 25. Peaks from the XRPD pattern arelisted in Table 18.

In some embodiments, crystalline Form IX is characterized by an XRPDpattern comprising a peak, in terms of 2θ, at 13.8°±0.2°. In someembodiments, crystalline Form IX has an XRPD pattern comprising thefollowing peaks, in terms of 2θ: 13.8°±0.2°; 17.4°±0.2°; 22.8°±0.2°; and24.8°±0.2°. In some embodiments, crystalline Form IX has an XRPD patterncomprising 2 or more, 3 or more, or 4 or more of the following peaks, interms of 2θ: 11.1°±0.2°; 12.2°±0.2°; 13.8°±0.2°; 15.5°±0.2°; 17.4°±0.2°;19.3°±0.2°; 20.9°±0.2°; 22.4°±0.2°; 22.8°±0.2°; and 24.8°±0.2°.

In some embodiments, Form IX is characterized by a DSC thermogramcomprising an endothermic peak having a maximum at about 256° C. In someembodiments, crystalline Form IX is characterized by the DSC thermogramsubstantially as shown in FIG. 26.

In some embodiments, crystalline Form IX has a TGA trace substantiallyas shown in FIG. 27.

Crystalline Form X

In some embodiments, the crystalline form of the compound of Formula Iis Form X. In some embodiments, Form X can be prepared by a processcomprising combining Form I with acetone. In some embodiments, theprocess further comprises heating the mixture resulting from thecombining of crystalline Form I and acetone. In some embodiments, themixture is heated to about 30 to about 70° C., about 40 to about 60° C.,or about 50° C. to yield Form X.

Crystalline Form X can be identified by unique signatures with respectto, for example, XRPD, DSC, and TGA. In some embodiments, crystallineForm X is characterized by an XRPD pattern substantially as shown inFIG. 28. Peaks from the XRPD pattern are listed in Table 19.

In some embodiments, crystalline Form X is characterized by an XRPDpattern comprising a peak, in terms of 2θ, at 6.8°±0.2°. In someembodiments, crystalline Form X has an XRPD pattern comprising thefollowing peaks, in terms of 2θ: 6.8°±0.2°; 13.5°±0.2°; 14.8°±0.2°; and17.0°±0.2°. In some embodiments, crystalline Form X has an XRPD patterncomprising 2 or more, 3 or more, or 4 or more of the following peaks, interms of 2θ: 6.8°±0.2°; 13.5°±0.2°; 14.8°±0.2°; 17.0°±0.2°; 19.6°±0.2°;20.2°±0.2°; 21.7°±0.2°; 25.0°±0.2°; and 26.3°±0.2°.

In some embodiments, Form X is characterized by a DSC thermogramcomprising an endothermic peak having a maximum at about 258° C. In someembodiments, crystalline Form X is characterized by the DSC thermogramsubstantially as shown in FIG. 29.

In some embodiments, crystalline Form X has a TGA trace substantially asshown in FIG. 30.

Crystalline Form XI

In some embodiments, the crystalline form of the compound of Formula Iis Form XI. Crystalline Form XI can be prepared by combining Form I withtetrahydrofuran or isobutyl acetate. The resulting mixture can beoptionally heated.

Crystalline Form XI can be identified by unique signatures with respectto, for example, XRPD and DSC. In some embodiments, crystalline Form XIis characterized by an XRPD pattern substantially as shown in FIG. 31.Peaks from the XRPD pattern are listed in Table 20.

In some embodiments, crystalline Form XI is characterized by an XRPDpattern comprising a peak, in terms of 2θ, at 7.7°±0.2°. In someembodiments, crystalline Form XI has an XRPD pattern comprising thefollowing peaks, in terms of 2θ: 7.7°±0.2°; 20.3°±0.2°; and 23.4°±0.2°.In some embodiments, crystalline Form XI has an XRPD pattern comprising2 or more, 3 or more, or 4 or more of the following peaks, in terms of2θ: 7.7°±0.2°; 12.4°±0.2°; 16.6°±0.2°; 20.3°±0.2°; 23.4°±0.2°;24.2°±0.2°; 26.3°±0.2°; and 29.9°±0.2°.

In some embodiments, Form IX is characterized by a DSC thermogramcomprising an endothermic peak having a maximum at about 117° C. In someembodiments, crystalline Form XI is characterized by the DSC thermogramsubstantially as shown in FIG. 32 (upper).

Crystalline Form XII

In some embodiments, the crystalline form of the compound of Formula Iis Form XII. Crystalline Form XII can be prepared by combining Form Iwith methyl t-butyl ether. The preparation method can optionally furthercomprise heating the resulting mixture.

Crystalline Form XII can be identified by unique signatures with respectto, for example, XRPD and DSC. In some embodiments, crystalline Form XIIis characterized by an XRPD pattern substantially as shown in FIG. 33.Peaks from the XRPD pattern are listed in Table 21.

In some embodiments, crystalline Form XII is characterized by an XRPDpattern comprising a peak, in terms of 2θ, at 7.8°±0.2°. In someembodiments, crystalline Form XII has an XRPD pattern comprising thefollowing peaks, in terms of 2θ: 7.8°±0.2°; 18.2°±0.2°; 20.1°±0.2°; and23.7°±0.2°. In some embodiments, crystalline Form XII has an XRPDpattern comprising 4 or more of the following peaks, in terms of 2θ:7.8°±0.2°; 12.6°±0.2°; 16.6°±0.2°; 18.2°±0.2°; 20.1°±0.2°; and23.7°±0.2°.

In some embodiments, Form XII is characterized by a DSC thermogramcomprising an endothermic peak having a maximum at about 137° C. In someembodiments, crystalline Form XII is characterized by the DSC tracesubstantially as shown in FIG. 34.

Crystalline Form XIII

In some embodiments, the crystalline form of the compound of Formula Iis Form XIII Crystalline Form XIII of the compound of Formula I can beprepared by combining Form I with ethanol or 2-methoxyethanol, ormixtures thereof. The preparation method can optionally further compriseheating the resulting mixture.

Crystalline Form XIII can be identified by unique signatures withrespect to, for example, XRPD and DSC. For example, in some embodiments,crystalline Form XIII is characterized by an XRPD pattern substantiallyas shown in FIG. 35. Peaks from the XRPD pattern are listed in Table 22.

In some embodiments, crystalline Form XIII is characterized by an XRPDpattern comprising a peak, in terms of 2θ, at 5.9°±0.2°. In someembodiments, crystalline Form XIII has an XRPD pattern comprising thefollowing peaks, in terms of 2θ: 5.9°±0.2°; 10.1°±0.2°; 13.0°±0.2°; and16.9°±0.2°. In some embodiments, crystalline Form XIII has an XRPDpattern comprising 2 or more, 3 or more, or 4 or more of the followingpeaks, in terms of 2θ: 3.8°±0.2°; 5.9° k 0.2°; 10.1°±0.2°; 13.0°±0.2°;16.9°±0.2°; 23.4°±0.2°; 26.0°±0.2°; and 26.9°±0.2°.

In some embodiments, crystalline Form XIII is characterized by the DSCthermogram substantially as shown in FIG. 36.

Methods

The crystalline forms of the invention can modulate activity of one ormore of various kinases including, for example, phosphoinositide3-kinases (PI3Ks). The term “modulate” is meant to refer to an abilityto increase or decrease the activity of one or more members of the PI3Kfamily. Accordingly, the crystalline forms of the invention can be usedin methods of modulating a PI3K by contacting the PI3K with any one ormore of the crystalline forms or compositions described herein. In someembodiments, crystalline forms of the present invention can act asinhibitors of one or more PI3Ks. In further embodiments, the compoundsof the invention can be used to modulate activity of a PI3K in anindividual in need of modulation of the receptor by administering amodulating amount of a crystalline form of the invention, or apharmaceutically acceptable salt thereof. In some embodiments,modulating is inhibiting.

Given that cancer cell growth and survival is impacted by multiplesignaling pathways, the present invention is useful for treating diseasestates characterized by drug resistant kinase mutants. In addition,different kinase inhibitors, exhibiting different preferences in thekinases which they modulate the activities of, may be used incombination. This approach could prove highly efficient in treatingdisease states by targeting multiple signaling pathways, reduce thelikelihood of drug-resistance arising in a cell, and reduce the toxicityof treatments for disease.

Kinases to which the present crystalline forms bind and/or modulate(e.g., inhibit) include any member of the PI3K family. In someembodiments, the PI3K is PI3Kα, PI3Kβ, PI3Kγ, or PI3Kδ. In someembodiments, the PI3K is PI3Kγ or PI3Kδ. In some embodiments, the PI3Kis PI3Kγ. In some embodiments, the PI3K is PI3Kδ. In some embodiments,the PI3K includes a mutation. A mutation can be a replacement of oneamino acid for another, or a deletion of one or more amino acids. Insuch embodiments, the mutation can be present in the kinase domain ofthe PI3K.

In some embodiments, more than one crystalline form of the invention isused to inhibit the activity of one kinase (e.g., PI3Kγ or PI3Kδ).

In some embodiments, more than one crystalline form of the invention isused to inhibit more than one kinase, such as at least two kinases(e.g., PI3Kγ and PI3Kδ).

In some embodiments, one or more of the crystalline forms is used incombination with another kinase inhibitor to inhibit the activity of onekinase (e.g., PI3Kγ or PI3Kδ).

In some embodiments, one or more of the crystalline forms is used incombination with another kinase inhibitor to inhibit the activities ofmore than one kinase (e.g., PI3Kγ or PI3Kδ), such as at least twokinases.

Another aspect of the present invention pertains to methods of treatinga kinase (such as PI3K)-associated disease or disorder in an individual(e.g., patient) by administering to the individual in need of suchtreatment a therapeutically effective amount or dose of one or morecrystalline forms of the present invention or a pharmaceuticalcomposition thereof. A PI3K-associated disease can include any disease,disorder or condition that is directly or indirectly linked toexpression or activity of the PI3K, including overexpression and/orabnormal activity levels. In some embodiments, the disease can be linkedto Akt (protein kinase B), mammalian target of rapamycin (mTOR), orphosphoinositide-dependent kinase 1 (PDK1). In some embodiments, themTOR-related disease can be inflammation, atherosclerosis, psoriasis,restenosis, benign prostatic hypertrophy, bone disorders, pancreatitis,angiogenesis, diabetic retinopathy, arthritis, immunological disorders,kidney disease, or cancer. A PI3K-associated disease can also includeany disease, disorder or condition that can be prevented, ameliorated,or cured by modulating PI3K activity. In some embodiments, the diseaseis characterized by the abnormal activity of PI3K. In some embodiments,the disease is characterized by mutant PI3K. In such embodiments, themutation can be present in the kinase domain of the PI3K.

Examples of PI3K-associated diseases include immune-based diseasesinvolving the system including, for example, rheumatoid arthritis,allergy, asthma, glomerulonephritis, lupus, or inflammation related toany of the above.

Further examples of PI3K-associated diseases include cancers such asbreast, prostate, colon, endometrial, brain, bladder, skin, uterus,ovary, lung, pancreatic, renal, gastric, or hematological cancer.

In some embodiments, the hematological cancer is acute myeloblasticleukemia (AML) or chronic myeloid leukemia (CIVIL), or B cell lymphoma.

Further examples of PI3K-associated diseases include lung diseases suchas acute lung injury (ALI) and adult respiratory distress syndrome(ARDS).

Further examples of PI3K-associated diseases include osteoarthritis,restenosis, atherosclerosis, bone disorders, arthritis, diabeticretinopathy, psoriasis, benign prostatic hypertrophy, inflammation,angiogenesis, pancreatitis, kidney disease, inflammatory bowel disease,myasthenia gravis, multiple sclerosis, or Sjögren's syndrome, and thelike.

Further examples of PI3K-associated diseases include idiopathicthrombocytopenic purpura (ITP), autoimmune hemolytic anemia (AIHA),vasculitis, systemic lupus erythematosus, lupus nephritis, pemphigus,membranous nephropathy, chronic lymphocytic leukemia (CLL), Non-Hodgkinlymphoma, hairy cell leukemia, Mantle cell lymphoma, Burkitt lymphoma,small lymphocytic lymphoma, follicular lymphoma, lymphoplasmacyticlymphoma, extranodal marginal zone lymphoma, activated B-cell like (ABC)diffuse large B cell lymphoma, or germinal center B cell (GCB) diffuselarge B cell lymphoma.

In some embodiments, the present application provides a method oftreating pemphigus, membranous nephropathy, Hodgkin's lymphoma,Waldenstrom's macroglobulinemia, prolymphocytic leukemia, acutelymphoblastic leukemia, myelofibrosis, mucosa-associated lymphatictissue (MALT) lymphoma, mediastinal (thymic) large B-cell lymphoma,lymphomatoid granulomatosis, splenic marginal zone lymphoma, primaryeffusion lymphoma, intravascular large B-cell lymphoma, plasma cellleukemia, extramedullary plasmacytoma, smouldering myeloma (akaasymptomatic myeloma), or monoclonal gammopathy of undeterminedsignificance (MGUS).

In some embodiments, the present application provides a method oftreating osteoarthritis, restenosis, atherosclerosis, bone disorders,arthritis, diabetic retinopathy, psoriasis, benign prostatichypertrophy, inflammation, angiogenesis, pancreatitis, kidney disease,inflammatory bowel disease, myasthenia gravis, multiple sclerosis, orSjögren's syndrome.

In some embodiments, the disease is idiopathic thrombocytopenic purpura(ITP), autoimmune hemolytic anemia (AIHA), vasculitis, pemphigus, ormembranous nephropathy.

In some embodiments, the idiopathic thrombocytopenic purpura (ITP) isselected from relapsed ITP and refractory ITP.

In some embodiments, the vasculitis is selected from Behçet's disease,Cogan's syndrome, giant cell arteritis, polymyalgia rheumatica (PMR),Takayasu's arteritis, Buerger's disease (thromboangiitis obliterans),central nervous system vasculitis, Kawasaki disease, polyarteritisnodosa, Churg-Strauss syndrome, mixed cryoglobulinemia vasculitis(essential or hepatitis C virus (HCV)-induced), Henoch-Schönlein purpura(HSP), hypersensitivity vasculitis, microscopic polyangiitis, Wegener'sgranulomatosis, and anti-neutrophil cytoplasm antibody associated (ANCA)systemic vasculitis (AASV).

In some embodiments, the present application provides methods oftreating an immune-based disease, cancer, or lung disease in a patient.

In some embodiments, the immune-based disease is systemic lupuserythematosus or lupus nephritis.

In some embodiments, the cancer is breast cancer, prostate cancer, coloncancer, endometrial cancer, brain cancer, bladder cancer, skin cancer,cancer of the uterus, cancer of the ovary, lung cancer, pancreaticcancer, renal cancer, gastric cancer, or a hematological cancer.

In some embodiments, the hematological cancer is acute myeloblasticleukemia, chronic myeloid leukemia, B cell lymphoma, chronic lymphocyticleukemia (CLL), Non-Hodgkins lymphoma, hairy cell leukemia, Mantle celllymphoma, Burkitt lymphoma, small lymphocytic lymphoma, follicularlymphoma, lymphoplasmacytic lymphoma, extranodal marginal zone lymphoma,activated B-cell like (ABC) diffuse large B cell lymphoma, or germinalcenter B cell (GCB) diffuse large B cell lymphoma.

In some embodiments, the non-Hodgkin lymphoma (NHL) is selected fromrelapsed NHL, refractory NHL, and recurrent follicular NHL.

In some embodiments, the lung disease is acute lung injury (ALI) oradult respiratory distress syndrome (ARDS).

As used herein, the term “contacting” refers to the bringing together ofindicated moieties in an in vitro system or an in vivo system. Forexample, “contacting” a PI3K with a crystalline form of the inventionincludes the administration of a crystalline form of the presentinvention to an individual or patient, such as a human, having a PI3K,as well as, for example, introducing a crystalline form of the inventioninto a sample containing a cellular or purified preparation containingthe PI3K.

As used herein, the term “individual” or “patient,” usedinterchangeably, refers to any animal, including mammals, preferablymice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep,horses, or primates, and most preferably humans.

As used herein, the phrase “therapeutically effective amount” refers tothe amount of active crystalline form or pharmaceutical agent thatelicits the biological or medicinal response that is being sought in atissue, system, animal, individual or human by a researcher,veterinarian, medical doctor or other clinician.

As used herein, the term “treating” or “treatment” refers to one or moreof (1) preventing the disease; for example, preventing a disease,condition or disorder in an individual who may be predisposed to thedisease, condition or disorder but does not yet experience or displaythe pathology or symptomatology of the disease; (2) inhibiting thedisease; for example, inhibiting a disease, condition or disorder in anindividual who is experiencing or displaying the pathology orsymptomatology of the disease, condition or disorder (i.e., arrestingfurther development of the pathology and/or symptomatology); and (3)ameliorating the disease; for example, ameliorating a disease, conditionor disorder in an individual who is experiencing or displaying thepathology or symptomatology of the disease, condition or disorder (i.e.,reversing the pathology and/or symptomatology) such as decreasing theseverity of disease.

Combination Therapies

One or more additional pharmaceutical agents such as, for example,chemotherapeutics, anti-inflammatory agents, steroids,immunosuppressants, as well as Bcr-Abl, Flt-3, EGFR, HER2, JAK (e.g.,JAK1 or JAK2), c-MET, VEGFR, PDGFR, cKit, IGF-1R, RAF, FAK, Akt mTOR,PIM, and AKT (e.g., AKT1, AKT2, or AKT3) kinase inhibitors such as, forexample, those described in WO 2006/056399, or other agents such as,therapeutic antibodies can be used in combination with the crystallineforms of the present invention for treatment of PI3K-associateddiseases, disorders or conditions. The one or more additionalpharmaceutical agents can be administered to a patient simultaneously orsequentially.

In some embodiments, the additional pharmaceutical agent is a JAK1and/or JAK2 inhibitor. In some embodiments, the present applicationprovides a method of treating a disease described herein (e.g., a B cellmalignancy, such as diffuse B-cell lymphoma) in a patient comprisingadministering to the patient a compound described herein, or apharmaceutically acceptable salt thereof, and a JAK1 and/or JAK2inhibitor. The B cell malignancies can include those described hereinand in U.S. Ser. No. 61/976,815, filed Apr. 8, 2014. In someembodiments, the inhibitor of JAK1 and/or JAK2 is3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile.In some embodiments, the inhibitor of JAK1 and/or JAK2 is(3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile(ruxolitinib; also known as INCB018424). Ruxolitinib has an IC₅₀ of lessthan 10 nM at 1 mM ATP (Assay D) at JAK1 and JAK2.3-Cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrileand ruxolitinib can be made by the procedure described in U.S. Pat. No.7,598,257 (Example 67), filed Dec. 12, 2006, which is incorporatedherein by reference in its entirety. In some embodiments, the inhibitorof JAK1 and/or JAK2 is(3R)-3-cyclopentyl-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrilephosphoric acid salt.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is a compound ofTable A, or a pharmaceutically acceptable salt thereof. The compounds inTable A are selective JAK1 inhibitors (selective over JAK2, JAK3, andTYK2). The IC₅₀s obtained by the method of Assay D at 1 mM ATP are shownin Table A.

TABLE A JAK1 IC₅₀ JAK2/ # Name/Reference Structure (nM) JAK1 1((2R,5S)-5-{2-[(1R)-1-hydroxyethyl]- 1H-imidazo[4,5-d]thieno[3,2-b]pyridin-1-yl}tetrahydro-2H-pyran- 2-yl)acetonitrile US 2014/0121198,Example 20

++ >10 2 4-[3-(cyanomethyl)-3-(3′,5′-dimethyl- 1H,1′H-4,4′-bipyrazol-1-yl)azetidin-1- yl]-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide US 2014/0343030, Example 7

+++ >10 3 3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile US 2010/0298334 Example 2^(a)

+ >10 4 3-(1-[1,3]oxazolo[5,4-b]pyridin-2- ylpyrrolidin-3-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H- pyrazol-1-yl]propanenitrile US2010/0298334 (Example 13c)

+ >10 5 4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1- yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile US 2011/0059951 (Example 12)

+ >10 6 4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrrol-1- yl]propyl}piperazin-1-yl)carbonyl]-3-flurobenzonitrile US 2011/0059951 (Example 13)

+ >10 7 {1-{1-[3-Fluoro-2- (trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3- d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile US 2011/0224190 (Example 1)

+ >10 8 4-{3-(Cyanomethyl)-3-[4-(7H- pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N-[4- fluoro-2-(trifluoromethyl)phenyl]piperidine-1- carboxamide US 2011/0224190(Example 154)

+ >10 9 [3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]-1-(1-{[2- (trifluoromethyl)pyrimidin-4-yl]carbonyl}piperidin-4-yl)azetidin-3- yl]acetonitrile US 2011/0224190(Example 85)

+ >10 10 [trans-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]- 3-(4-{[2-(trifluoromethyl)pyrimidin-4-yl]carbonyl}piperazin-1- yl)cyclobutyl]acetonitrile US 2012/0149681(Example 7b)

+ >10 11 {trans-3-(4-{[4-[(3-hydroxyazetidin- 1-yl)methyl]-6-(trifluoromethyl)pyridin-2- yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H- pyrazol-1-yl]cyclobutyl}acetonitrileUS 2012/0149681 (Example 157)

+ >10 12 {trans-3-(4-{[4-{[(2S)-2- (hydroxymethyl)pyrrolidin-1-yl]methyl}-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H- pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile US 2012/0149681 (Example 161)

+ >10 13 {trans-3-(4-{[4-{[(2R)-2- (hydroxymethyl)pyrrolidin-1-yl]methyl}-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H- pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile US 2012/0149681 (Example 162)

+ >10 14 4-(4-{3-[(dimethylamino)methyl]-5-fluorophenoxy}piperidin-1-yl)-3-[4- (7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]butanenitrile US 2012/0149682 (Example 20)^(b)

+ >10 15 5-{3-(cyanomethyl)-3-[4-(7H- pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N- isopropylpyrazine-2-carboxamide US2013/0018034 (Example 18)

+ >10 16 4-{3-(cyanomethyl)-3-[4-(7H- pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5- difluoro-N-[(1S)-2,2,2,-trifluoro-1-methylethyl]benzamide US 2013/0018034 (Example 28)

+ >10 17 5-{3-(cyanomethyl)-3-[4-(1H- pyrrolo[2,3-b]pyridin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-N- isopropylpyrazine-2-carboxamide US2013/0018034 (Example 34)

+ >10 18 {1-(cis-4-{[6-(2-hydroxyethyl)-2- (trifluoromethyl)pyrimidin-4-yl]oxy}cyclohexyl)-3-[4-(7H- pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile US 2013/0045963 (Example 45)

+ >10 19 {1-(cis-4-{[4-[(ethylamino)methyl]-6-(trifluoromethyl)pyridin-2- yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile US 2013/0045963 (Example 65)

+ >10 20 {1-(cis-4-{[4-(1-hydroxy-1- methylethyl)-6-(trifluoromethyl)pyridin-2- yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile US 2013/0045963 (Example 69)

+ >10 21 {1-(cis-4-{[4-{[(3R)-3- hydroxypyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2- yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile US 2013/0045963 (Example 95)

+ >10 22 {1-(cis-4-{[4-{[(3S)-3- hydroxypyrrolidin-1-yl]methyl}-6-(trifluoromethyl)pyridin-2- yl]oxy}cyclohexyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile US 2013/0045963 (Example 95)

+ >10 23 {trans-3-(4-{[4-({[(1S)-2-hydroxy-1-methylethyl]amino}methyl)-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H- pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile US 2014/0005166 (Example 1)

+ >10 24 {trans-3-(4-{[4-({[(2R)-2- hydroxypropyl]amino}methyl)-6-(trifluoromethyl)pyridin-2- yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H- pyrazol-1-yl]cyclobutyl}acetonitrileUS 2014/0005166 (Example 14)

+ >10 25 {trans-3-(4-{[4-({[(2S)-2- hydroxypropyl]amino}methyl)-6-(trifluoromethyl)pyridin-2- yl]oxy}piperidin-1-yl)-1-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H- pyrazol-1-yl]cyclobutyl}acetonitrileUS 2014/0005166 (Example 15)

+ >10 26 {trans-3-(4-{[4-(2-hydroxyethyl)-6- (trifluoromethyl)pyridin-2-yl]oxy}piperidin-1-yl)-1-[4-(7H- pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]cyclobutyl}acetonitrile US 2014/0005166 (Example 20)

+ >10 + means <10 nM (see Example D for assay conditions) ++ means ≤100nM (see Example D for assay conditions) +++ means ≤300 nM (see Example Dfor assay conditions) ^(a)Data for enantiomer 1 ^(b)Data for enantiomer2

In some embodiments, the inhibitor of JAK1 and/or JAK2 is{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrile,or a pharmaceutically acceptable salt thereof.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is{1-{1-[3-fluoro-2-(trifluoromethyl)isonicotinoyl]piperidin-4-yl}-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-3-yl}acetonitrileadipic acid salt.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is4-{3-(cyanomethyl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]azetidin-1-yl}-2,5-difluoro-N-[(1S)-2,2,2-trifluoro-1-methylethyl]benzamide,or a pharmaceutically acceptable salt thereof.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is selected from(R)-3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile,(R)-3-(1-[1,3]oxazolo[5,4-b]pyridin-2-ylpyrrolidin-3-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile,(R)-4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile,(R)-4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrrol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile,or(R)-4-(4-{3-[(dimethylamino)methyl]-5-fluorophenoxy}piperidin-1-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]butanenitrile,(S)-3-[1-(6-chloropyridin-2-yl)pyrrolidin-3-yl]-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile,(S)-3-(1-[1,3]oxazolo[5,4-b]pyridin-2-ylpyrrolidin-3-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propanenitrile,(S)-4-[(4-{3-cyano-2-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile,(S)-4-[(4-{3-cyano-2-[3-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrrol-1-yl]propyl}piperazin-1-yl)carbonyl]-3-fluorobenzonitrile,(S)-4-(4-{3-[(dimethylamino)methyl]-5-fluorophenoxy}piperidin-1-yl)-3-[4-(7H-pyrrolo[2,3-d]pyrimidin-4-yl)-1H-pyrazol-1-yl]butanenitrile;and pharmaceutically acceptable salts of any of the aforementioned.

In some embodiments, the compounds of Table A are prepared by thesynthetic procedures described in US Patent Publ. No. 2010/0298334,filed May 21, 2010, US Patent Publ. No. 2011/0059951, filed Aug. 31,2010, US Patent Publ. No. 2011/0224190, filed Mar. 9, 2011, US PatentPubl. No. 2012/0149681, filed Nov. 18, 2011, US Patent Publ. No.2012/0149682, filed Nov. 18, 2011, US Patent Publ. 2013/0018034, filedJun. 19, 2012, US Patent Publ. No. 2013/0045963, filed Aug. 17, 2012,and US Patent Publ. No. 2014/0005166, filed May 17, 2013, each of whichis incorporated herein by reference in its entirety.

In some embodiments, the inhibitor of JAK1 and/or JAK2 is selected fromthe compounds of US Patent Publ. No. 2010/0298334, filed May 21, 2010,US Patent Publ. No. 2011/0059951, filed Aug. 31, 2010, US Patent Publ.No. 2011/0224190, filed Mar. 9, 2011, US Patent Publ. No. 2012/0149681,filed Nov. 18, 2011, US Patent Publ. No. 2012/0149682, filed Nov. 18,2011, US Patent Publ. 2013/0018034, filed Jun. 19, 2012, US Patent Publ.No. 2013/0045963, filed Aug. 17, 2012, and US Patent Publ. No.2014/0005166, filed May 17, 2013, each of which is incorporated hereinby reference in its entirety.

Example antibodies for use in combination therapy include but are notlimited to Trastuzumab (e.g. anti-HER2), Ranibizumab (e.g. anti-VEGF-A),Bevacizumab (trade name Avastin, e.g. anti-VEGF, Panitumumab (e.g.anti-EGFR), Cetuximab (e.g. anti-EGFR), Rituxan (anti-CD20) andantibodies directed to c-MET.

One or more of the following agents may be used in combination with thecrystalline forms of the present invention and are presented as a nonlimiting list: a cytostatic agent, cisplatin, doxorubicin, taxotere,taxol, etoposide, irinotecan, camptostar, topotecan, paclitaxel,docetaxel, epothilones, tamoxifen, 5-fluorouracil, methoxtrexate,temozolomide, cyclophosphamide, SCH 66336, R115777, L778,123, BMS214662, Iressa, Tarceva, antibodies to EGFR, Gleevec™, intron, ara-C,adriamycin, cytoxan, gemcitabine, Uracil mustard, Chlormethine,Ifosfamide, Melphalan, Chlorambucil, Pipobroman, Triethylenemelamine,Triethylenethiophosphoramine, Busulfan, Carmustine, Lomustine,Streptozocin, Dacarbazine, Floxuridine, Cytarabine, 6-Mercaptopurine,6-Thioguanine, Fludarabine phosphate, oxaliplatin, leucovirin,ELOXATIN™, Pentostatine, Vinblastine, Vincristine, Vindesine, Bleomycin,Dactinomycin, Daunorubicin, Doxorubicin, Epirubicin, Idarubicin,Mithramycin, Deoxycoformycin, Mitomycin-C, L-Asparaginase, Teniposide17.alpha.-Ethinylestradiol, Diethylstilbestrol, Testosterone,Prednisone, Fluoxymesterone, Dromostanolone propionate, Testolactone,Megestrolacetate, Methylprednisolone, Methyltestosterone, Prednisolone,Triamcinolone, Chlorotrianisene, Hydroxyprogesterone, Aminoglutethimide,Estramustine, Medroxyprogesteroneacetate, Leuprolide, Flutamide,Toremifene, goserelin, Cisplatin, Carboplatin, Hydroxyurea, Amsacrine,Procarbazine, Mitotane, Mitoxantrone, Levamisole, Navelbene,Anastrazole, Letrazole, Capecitabine, Reloxafine, Droloxafine,Hexamethylmelamine, Avastin, herceptin, Bexxar, Velcade, Zevalin,Trisenox, Xeloda, Vinorelbine, Porfimer, Erbitux, Liposomal, Thiotepa,Altretamine, Melphalan, Trastuzumab, Lerozole, Fulvestrant, Exemestane,Fulvestrant, Ifosfomide, Rituximab, C225, Campath, Clofarabine,cladribine, aphidicolon, rituxan, sunitinib, dasatinib, tezacitabine,Sml1, fludarabine, pentostatin, triapine, didox, trimidox, amidox, 3-AP,MDL-101,731, bendamustine (Treanda), ofatumumab, and GS-1101 (also knownas CAL-101).

Example chemotherapeutics include proteosome inhibitors (e.g.,bortezomib), thalidomide, revlimid, and DNA-damaging agents such asmelphalan, doxorubicin, cyclophosphamide, vincristine, etoposide,carmustine, and the like.

Example steroids include corticosteroids such as dexamethasone orprednisone.

Example Bcr-Abl inhibitors include the compounds, and pharmaceuticallyacceptable salts thereof, of the genera and species disclosed in U.S.Pat. No. 5,521,184, WO 04/005281, and U.S. Ser. No. 60/578,491.

Example suitable Flt-3 inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 03/037347, WO03/099771, and WO 04/046120.

Example suitable RAF inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 00/09495 and WO05/028444.

Example suitable FAK inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 04/080980, WO04/056786, WO 03/024967, WO 01/064655, WO 00/053595, and WO 01/014402.

Example suitable mTOR inhibitors include compounds, and theirpharmaceutically acceptable salts, as disclosed in WO 2011/025889.

In some embodiments, the crystalline forms of the invention can be usedin combination with one or more other kinase inhibitors includingimatinib, particularly for treating patients resistant to imatinib orother kinase inhibitors.

In some embodiments, the crystalline forms of the invention can be usedin combination with a chemotherapeutic in the treatment of cancer, suchas multiple myeloma, and may improve the treatment response as comparedto the response to the chemotherapeutic agent alone, withoutexacerbation of its toxic effects. Examples of additional pharmaceuticalagents used in the treatment of multiple myeloma, for example, caninclude, without limitation, melphalan, melphalan plus prednisone [MP],doxorubicin, dexamethasone, and Velcade (bortezomib). Further additionalagents used in the treatment of multiple myeloma include Bcr-Abl, Flt-3,RAF and FAK kinase inhibitors. Additive or synergistic effects aredesirable outcomes of combining a PI3K inhibitor of the presentinvention with an additional agent. Furthermore, resistance of multiplemyeloma cells to agents such as dexamethasone may be reversible upontreatment with the PI3K inhibitor of the present invention. The agentscan be combined with the present crystalline form in a single orcontinuous dosage form, or the agents can be administered simultaneouslyor sequentially as separate dosage forms.

In some embodiments, a corticosteroid such as dexamethasone isadministered to a patient in combination with the crystalline forms ofthe invention where the dexamethasone is administered intermittently asopposed to continuously.

In some further embodiments, combinations of the crystalline forms ofthe invention with other therapeutic agents can be administered to apatient prior to, during, and/or after a bone marrow transplant or stemcell transplant.

Pharmaceutical Formulations and Dosage Forms

When employed as pharmaceuticals, the crystalline forms of the inventioncan be administered in the form of pharmaceutical compositions. Thesecompositions can be prepared in a manner well known in thepharmaceutical art, and can be administered by a variety of routes,depending upon whether local or systemic treatment is desired and uponthe area to be treated. Administration may be topical (includingtransdermal, epidermal, ophthalmic and to mucous membranes includingintranasal, vaginal and rectal delivery), pulmonary (e.g., by inhalationor insufflation of powders or aerosols, including by nebulizer;intratracheal or intranasal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal intramuscular or injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration. Parenteraladministration can be in the form of a single bolus dose, or may be, forexample, by a continuous perfusion pump. Pharmaceutical compositions andformulations for topical administration may include transdermal patches,ointments, lotions, creams, gels, drops, suppositories, sprays, liquidsand powders. Conventional pharmaceutical carriers, aqueous, powder oroily bases, thickeners and the like may be necessary or desirable.

This invention also includes pharmaceutical compositions which contain,as the active ingredient, the crystalline form of the invention incombination with one or more pharmaceutically acceptable carriers(excipients). In some embodiments, the composition is suitable fortopical administration. In making the compositions of the invention, theactive ingredient is typically mixed with an excipient, diluted by anexcipient or enclosed within such a carrier in the form of, for example,a capsule, sachet, paper, or other container. When the excipient servesas a diluent, it can be a solid, semi-solid, or liquid material, whichacts as a vehicle, carrier or medium for the active ingredient. Thus,the compositions can be in the form of tablets, pills, powders,lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions,syrups, aerosols (as a solid or in a liquid medium), ointmentscontaining, for example, up to 10% by weight of the active crystallineform, soft and hard gelatin capsules, suppositories, sterile injectablesolutions, and sterile packaged powders.

In preparing a formulation, the active crystalline form can be milled toprovide the appropriate particle size prior to combining with the otheringredients. If the active crystalline form is substantially insoluble,it can be milled to a particle size of less than 200 mesh. If the activecrystalline form is substantially water soluble, the particle size canbe adjusted by milling to provide a substantially uniform distributionin the formulation, e.g. about 40 mesh.

The crystalline forms of the invention may be milled using known millingprocedures such as wet milling to obtain a particle size appropriate fortablet formation and for other formulation types. Finely divided(nanoparticulate) preparations of the crystalline forms of the inventioncan be prepared by processes known in the art, e.g., see InternationalApp. No. WO 2002/000196.

Some examples of suitable excipients include lactose, dextrose, sucrose,sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates,tragacanth, gelatin, calcium silicate, microcrystalline cellulose,polyvinylpyrrolidone, cellulose, water, syrup, and methyl cellulose. Theformulations can additionally include: lubricating agents such as talc,magnesium stearate, and mineral oil; wetting agents; emulsifying andsuspending agents; preserving agents such as methyl- andpropylhydroxy-benzoates; sweetening agents; and flavoring agents. Thecompositions of the invention can be formulated so as to provide quick,sustained or delayed release of the active ingredient afteradministration to the patient by employing procedures known in the art.

The compositions can be formulated in a unit dosage form, each dosagecontaining from about 5 to about 1000 mg (1 g), more usually about 100to about 500 mg, of the active ingredient. The term “unit dosage forms”refers to physically discrete units suitable as unitary dosages forhuman subjects and other mammals, each unit containing a predeterminedquantity of active material calculated to produce the desiredtherapeutic effect, in association with a suitable pharmaceuticalexcipient.

In some embodiments, the crystalline forms or compositions of theinvention contain from about 5 to about 50 mg of the active ingredient.One having ordinary skill in the art will appreciate that this embodiescrystalline forms or compositions containing about 5 to about 10, about10 to about 15, about 15 to about 20, about 20 to about 25, about 25 toabout 30, about 30 to about 35, about 35 to about 40, about 40 to about45, or about 45 to about 50 mg of the active ingredient.

In some embodiments, the crystalline forms or compositions of theinvention contain from about 50 to about 500 mg of the activeingredient. One having ordinary skill in the art will appreciate thatthis embodies crystalline forms or compositions containing about 50 toabout 100, about 100 to about 150, about 150 to about 200, about 200 toabout 250, about 250 to about 300, about 350 to about 400, or about 450to about 500 mg of the active ingredient.

In some embodiments, the crystalline forms or compositions of theinvention contain from about 500 to about 1000 mg of the activeingredient. One having ordinary skill in the art will appreciate thatthis embodies crystalline forms or compositions containing about 500 toabout 550, about 550 to about 600, about 600 to about 650, about 650 toabout 700, about 700 to about 750, about 750 to about 800, about 800 toabout 850, about 850 to about 900, about 900 to about 950, or about 950to about 1000 mg of the active ingredient.

Similar dosages may be used of the crystalline forms described herein inthe methods and uses of the invention.

The active crystalline form can be effective over a wide dosage rangeand is generally administered in a pharmaceutically effective amount. Itwill be understood, however, that the amount of the crystalline formactually administered will usually be determined by a physician,according to the relevant circumstances, including the condition to betreated, the chosen route of administration, the actual crystalline formadministered, the age, weight, and response of the individual patient,the severity of the patient's symptoms, and the like.

For preparing solid compositions such as tablets, the principal activeingredient is mixed with a pharmaceutical excipient to form a solidpreformulation composition containing a homogeneous mixture of acrystalline form of the present invention. When referring to thesepreformulation compositions as homogeneous, the active ingredient istypically dispersed evenly throughout the composition so that thecomposition can be readily subdivided into equally effective unit dosageforms such as tablets, pills and capsules. This solid preformulation isthen subdivided into unit dosage forms of the type described abovecontaining from, for example, about 0.1 to about 1000 mg of the activeingredient of the present invention.

The tablets or pills of the present invention can be coated or otherwisecompounded to provide a dosage form affording the advantage of prolongedaction. For example, the tablet or pill can comprise an inner dosage andan outer dosage component, the latter being in the form of an envelopeover the former. The two components can be separated by an enteric layerwhich serves to resist disintegration in the stomach and permit theinner component to pass intact into the duodenum or to be delayed inrelease. A variety of materials can be used for such enteric layers orcoatings, such materials including a number of polymeric acids andmixtures of polymeric acids with such materials as shellac, cetylalcohol, and cellulose acetate.

The liquid forms in which the crystalline forms and compositions of thepresent invention can be incorporated for administration orally or byinjection include aqueous solutions, suitably flavored syrups, aqueousor oil suspensions, and flavored emulsions with edible oils such ascottonseed oil, sesame oil, coconut oil, or peanut oil, as well aselixirs and similar pharmaceutical vehicles.

Compositions for inhalation or insufflation include solutions andsuspensions in pharmaceutically acceptable, aqueous or organic solvents,or mixtures thereof, and powders. The liquid or solid compositions maycontain suitable pharmaceutically acceptable excipients as describedsupra. In some embodiments, the compositions are administered by theoral or nasal respiratory route for local or systemic effect.Compositions in can be nebulized by use of inert gases. Nebulizedsolutions may be breathed directly from the nebulizing device or thenebulizing device can be attached to a face mask, tent, or intermittentpositive pressure breathing machine. Solution, suspension, or powdercompositions can be administered orally or nasally from devices whichdeliver the formulation in an appropriate manner.

Topical formulations can contain one or more conventional carriers. Insome embodiments, ointments can contain water and one or morehydrophobic carriers selected from, for example, liquid paraffin,polyoxyethylene alkyl ether, propylene glycol, white Vaseline, and thelike. Carrier compositions of creams can be based on water incombination with glycerol and one or more other components, e.g.glycerinemonostearate, PEG-glycerinemonostearate and cetylstearylalcohol. Gels can be formulated using isopropyl alcohol and water,suitably in combination with other components such as, for example,glycerol, hydroxyethyl cellulose, and the like. In some embodiments,topical formulations contain at least about 0.1, at least about 0.25, atleast about 0.5, at least about 1, at least about 2, or at least about 5wt % of the crystalline form of the invention. The topical formulationscan be suitably packaged in tubes of, for example, 100 g which areoptionally associated with instructions for the treatment of the selectindication, e.g., psoriasis or other skin condition.

The amount of crystalline form or composition administered to a patientwill vary depending upon what is being administered, the purpose of theadministration, such as prophylaxis or therapy, the state of thepatient, the manner of administration, and the like. In therapeuticapplications, compositions can be administered to a patient alreadysuffering from a disease in an amount sufficient to cure or at leastpartially arrest the symptoms of the disease and its complications.Effective doses will depend on the disease condition being treated aswell as by the judgment of the attending clinician depending uponfactors such as the severity of the disease, the age, weight and generalcondition of the patient, and the like.

The compositions administered to a patient can be in the form ofpharmaceutical compositions described above. These compositions can besterilized by conventional sterilization techniques, or may be sterilefiltered. Aqueous solutions can be packaged for use as is, orlyophilized, the lyophilized preparation being combined with a sterileaqueous carrier prior to administration. The pH of the crystalline formpreparations typically will be between 3 and 11, more preferably from 5to 9 and most preferably from 7 to 8. It will be understood that use ofcertain of the foregoing excipients, carriers, or stabilizers willresult in the formation of pharmaceutical salts.

The therapeutic dosage of a crystalline form of the present applicationcan vary according to, for example, the particular use for which thetreatment is made, the manner of administration of the crystalline form,the health and condition of the patient, and the judgment of theprescribing physician. The proportion or concentration of a crystallineform of the invention in a pharmaceutical composition can vary dependingupon a number of factors including dosage, chemical characteristics(e.g., hydrophobicity), and the route of administration. For example,the crystalline forms of the invention can be provided in an aqueousphysiological buffer solution containing about 0.1 to about 10% w/v ofthe crystalline form for parenteral administration. Some typical doseranges are from about 1 μg/kg to about 1 g/kg of body weight per day. Insome embodiments, the dose range is from about 0.01 mg/kg to about 100mg/kg of body weight per day. The dosage is likely to depend on suchvariables as the type and extent of progression of the disease ordisorder, the overall health status of the particular patient, therelative biological efficacy of the crystalline form selected,formulation of the excipient, and its route of administration. Effectivedoses can be extrapolated from dose-response curves derived from invitro or animal model test systems.

The compositions of the invention can further include one or moreadditional pharmaceutical agents such as a chemotherapeutic, steroid,anti-inflammatory compound, or immunosuppressant, examples of which arelisted hereinabove.

Labeled Compounds and Assay Methods

Another aspect of the present invention relates to labeled crystallineforms of the invention (radio-labeled, fluorescent-labeled, etc.) thatwould be useful not only in imaging techniques but also in assays, bothin vitro and in vivo, for localizing and quantitating PI3K in tissuesamples, including human, and for identifying PI3K ligands by inhibitionbinding of a labeled compound. Accordingly, the present inventionincludes PI3K assays that contain such labeled compounds.

The present invention further includes isotopically-labeled crystallineforms of the invention. An “isotopically” or “radio-labeled” crystallineform is a crystalline form of the invention where one or more atoms arereplaced or substituted by an atom having an atomic mass or mass numberdifferent from the atomic mass or mass number typically found in nature(i.e., naturally occurring). Suitable radionuclides that may beincorporated in crystalline forms of the present invention include butare not limited to ²H (also written as D for deuterium), ³H (alsowritten as T for tritium), ¹¹C, ¹³C, ¹⁴C, ¹⁵N, ¹⁵O, ¹⁷O, ¹⁸O, ¹⁸F, ³⁵S,³⁶Cl, ⁸²Br, ⁷⁵Br, ⁷⁶Br, ⁷⁷Br, ¹²³I, ¹²⁴I, ¹²⁵I and a ¹³¹I. Theradionuclide that is incorporated in the instant radio-labeledcrystalline form will depend on the specific application of thatradio-labeled crystalline form. For example, for in vitro PI3K labelingand competition assays, crystalline forms that incorporate ³H, ¹⁴C,⁸²Br, ¹²⁵I, ¹³¹I, ³⁵S or will generally be most useful. Forradio-imaging applications ¹¹C, ¹⁸F, ¹²⁵I, ¹²³I, ¹²⁴I, ¹³¹I, ⁷⁵Br, ⁷⁶Bror ⁷⁷Br will generally be most useful.

It is understood that a “radio-labeled” or “labeled compound” is acrystalline form that has incorporated at least one radionuclide. Insome embodiments the radionuclide is selected from the group consistingof ³H, ¹⁴C, ¹²⁵I, ³⁵S and ⁸²Br. In some embodiments, one or more H atomsfor any crystalline form described herein is each replaced by adeuterium atom.

The present invention can further include synthetic methods forincorporating radio-isotopes into crystalline forms of the invention.Synthetic methods for incorporating radio-isotopes into organiccompounds are well known in the art, and an ordinary skill in the artwill readily recognize the methods applicable for the crystalline formsof invention.

A labeled crystalline form of the invention can be used in a screeningassay to identify/evaluate compounds. For example, a newly synthesizedor identified compound (i.e., test compound) which is labeled can beevaluated for its ability to bind a PI3K by monitoring its concentrationvariation when contacting with the PI3K, through tracking of thelabeling. For example, a test compound (labeled) can be evaluated forits ability to reduce binding of another compound which is known to bindto a PI3K (i.e., standard compound). Accordingly, the ability of a testcompound to compete with the standard compound for binding to the PI3Kdirectly correlates to its binding affinity. Conversely, in some otherscreening assays, the standard compound is labeled and test compoundsare unlabeled. Accordingly, the concentration of the labeled standardcompound is monitored in order to evaluate the competition between thestandard compound and the test compound, and the relative bindingaffinity of the test compound is thus ascertained.

Kits

The present invention also includes pharmaceutical kits useful, forexample, in the treatment or prevention of PI3K-associated diseases ordisorders, such as cancer, which include one or more containerscontaining a pharmaceutical composition comprising a therapeuticallyeffective amount of a crystalline form of the invention. Such kits canfurther include, if desired, one or more of various conventionalpharmaceutical kit components, such as, for example, containers with oneor more pharmaceutically acceptable carriers, additional containers,etc., as will be readily apparent to those skilled in the art.Instructions, either as inserts or as labels, indicating quantities ofthe components to be administered, guidelines for administration, and/orguidelines for mixing the components, can also be included in the kit.

The invention will be described in greater detail by way of specificexamples. The following examples are offered for illustrative purposes,and are not intended to limit the invention in any manner. Those ofskill in the art will readily recognize a variety of non-criticalparameters which can be changed or modified to yield essentially thesame results.

EXAMPLES

In the below examples, X-Ray Powder Diffraction analysis was carried outon a Rigaku MiniFlex X-ray Powder Diffractometer (XRPD) instrument withthe following parameters: radiation source is Cu at 1.054056 Å withK_(β) filter and X-ray power of 30 KV, 15 mA. The sample powder wasdispersed on a zero-background sample holder. General measurementconditions were:

-   -   Start Angle—3°    -   Stop Angle—45°    -   Sampling—0.02 deg.    -   Scan speed—2 deg/min.

Differential Scanning calorimetry (DSC) was carried out on a TAInstrument Differential Scanning calorimetry, Model Q20 withautosampler. The general experimental conditions were: 30-260° C. at 10°C./min, nitrogen gas flow at 50 mL/min, using an aluminum sample pan.

Thermogravimetric analysis (TGA) was carried out on a TA InstrumentThermogravimetric Analyzer, Model Q500 with the following conditions:Ramp at 20° C./min. to 600° C.; nitrogen gas at 40 mL/min balance purgeflow; 60 mL/min sample purge flow; and platinum sample pan.

Dynamic Vapor Sorption (DVS) was performed in an SGA-100 Symmetric VaporSorption Analyzer from VTI Corporation. The moisture uptake profile wascompleted in three cycles in 10% RH increments with the first adsorptionfrom 25% to 95% RH, followed by desorption in 10% increments from 95% to5% RH. The equilibration criteria were 0.0050 wt % in 5 minutes with amaximum equilibration time of 180 minutes. All adsorption and desorptionwere performed at room temperature (25° C.). No pre-drying step wasapplied for the sample.

Example 1 Preparation and Characterization of Form I

A solution of concentrated HCl (141 mL, 1.69 mol, 1.2 eq.) in 2-propanol(1.51 L) was added to a solution of(S)-7-(1-((9H-purin-6-yl)amino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one(648 g, 92 wt %, 1.41 mol, 1.0 eq., see US Pat. Pub. No. 2011/0015212)in 2-propanol (7.1 L) under a nitrogen atmosphere in a reactor. Thereaction mixture was stirred at room temperature for about 25 minutes,then stirred at 79° C. for about 1.5 hours, and then stirred at roomtemperature for about 1 hour. The product was filtered, washed with2-propanol (3×0.55 L), washed with heptanes (3×0.55 L), and dried underreduced pressure to afford(S)-7-(1-((9H-purin-6-yl)amino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-onehydrochloride (550 g, 85% yield).

(S)-7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-onehydrochloride (325 g) and CH₂Cl₂ (3.5 L) were charged to a reactor undernitrogen. Aqueous Na₂CO₃ was charged until the pH was 12, and thereaction mixture was stirred for 40 minutes. The reaction mixture wasfiltered and the phases were separated. The aqueous phase and CH₂Cl₂(2.0 L) and conc. HCl (20 mL) were charged to a reactor and stirred for10 minutes until the pH was 2. Saturated aqueous K₂CO₃ (300 mL) wascharged until the pH was 12. The phases were separated, and the aqueousphase was extracted with CH₂Cl₂ (500 mL). The phases were separated, andthe organic phase was washed with brine (1000 mL) and dried over MgSO₄.The reaction mixture was filtered and the filter cake was washed withCH₂Cl₂ (2×300 mL). The combined organic phases were distilled underreduced pressure. Ethyl acetate (2.5 L) was charged to the reactor andthe distillation was continued at atmospheric pressure until thetemperature reached 68° C. The distillation was stopped, and thedistillation residue was cooled to 62° C. A 3:2 (v/v) mixture ofMeOH/CH₂Cl₂ (500 mL) was added and the reaction mixture was cooled toroom temperature. The reaction mixture was filtered, and the filter cakewas washed with chilled EtOAc (3×300 mL) and heptanes (3×300 mL) anddried under reduced pressure at 45-50° C. to afford(S)-7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one(Form I).

Form I was confirmed as a crystalline solid according to XRPD analysis.The XRPD pattern of Form I is shown in FIG. 1 and the peak data is givenbelow in Table 1.

TABLE 1 XRPD Peak Data for Form I. 2-Theta Height H % 9.2 85 7.6 10.01108 100 11.7 308 27.8 12.6 924 83.3 15.1 369 33.3 15.6 440 39.7 16.3285 25.7 16.7 100 9 18.0 745 67.2 19.1 72 6.5 19.9 319 28.8 20.3 37233.6 21.2 467 42.1 22.6 857 77.3 22.9 300 27.1 24.0 599 54.1 25.5 15213.7 25.9 374 33.7 26.8 228 20.6 27.6 157 14.2 28.0 420 37.9 29.0 31928.8 30.0 376 33.9 32.9 111 10 33.7 118 10.7 34.7 99 8.9

DSC analysis of Form I revealed one peak with an onset temperature of176° C. and a maximum at 183° C. The DSC thermogram is provided in FIG.2.

TGA analysis of Form I revealed 0.2% weight loss up to 100° C. The TGAthermogram is provided in FIG. 3.

Moisture adsorption/desorption of Form I was analyzed by DVS. Resultsfrom two DVS cycles are shown in FIG. 4. The data indicate that Form Iinitially contained about 0.3% water, and water adsorption increased to1.8% at 85% RH. The shapes of the isotherms indicate weakadsorbent-adsorbate interaction and a low moisture uptake at low vaporconcentration. Additionally, the data indicate a strong increase insorption at higher vapor concentration, with maximum adsorption at about85% RH.

Example 2 Crystalline Form Screening Methods and Results

New crystalline forms of the compound of Formula I were obtained fromthe various screening methods described below. Form I, as describedabove in Example I, was used as the starting material in the screensunless otherwise indicated.

Phase Equilibrium Screen at 25 and 50° C.

The compound of Formula I (from Example 1) was equilibrated in varioussolvents at 25+/−1° C. and 50+/−1° C. To 2 mL of saturated or cloudysolutions of the compound of Formula I prepared in various solvents, aslisted below in Tables 2 and 3, was added about 30 mg of additionalcompound of Formula I followed by stirring at 25±1° C. and at 50±1° C.The temperature was controlled by a IKA® ETS-D5 temperature controllerand a IKA® RCT basic safety control.

The supernatant was filtered and the excess solid phase was analyzed viaXRPD to determine crystallinity and the identity of any new crystallineforms. Results of the screens are indicated below in Tables 2 and 3. Theentry “N/A” means that either the sample contained only clear solutionor the amount of solid was too small to be analyzed by XRPD.

TABLE 2 Phase Equilibrium Results at 25° C. Solvent Form AcetonitrileForm I Chloroform N/A Methylene chloride N/A Dimethyl formamide N/A1,4-Dioxane Form VII Methanol N/A 2-Methoxyethanol N/A Methyl isobutylketone Form IX Toluene Form IV Hexane Form I Tetrahydrofuran N/A AcetoneForm I n-Butyl alcohol N/A Methyl t-butyl ether Amorphous Dimethylsulfoxide N/A Ethanol N/A Ethyl acetate Form I Ethyl formate Form IHeptane Form I Isobutyl acetate Form V Isopropyl acetate Form III1-Propanol N/A Isopropyl alcohol N/A Water Form VII Methyl ethyl ketoneForm I

TABLE 3 Phase Equilibrium Results at 50° C. Solvent Form AcetonitrileForm I Chloroform N/A Methylene chloride N/A Dimethyl formamide N/A1,4-Dioxane N/A Methanol N/A 2-Methoxyethanol N/A Methyl isobutyl ketoneForm IX Toluene Form IV Hexane Form I Tetrahydrofuran N/A Acetone Form Xn-Butyl alcohol N/A Methyl t-butyl ether Form XII Dimethyl sulfoxide N/AEthanol N/A Ethyl acetate Form I Ethyl formate Form I Heptane Form IIsobutyl acetate Form X Isopropyl acetate Form III 1-Propanol N/AIsopropyl alcohol Form II Water Form VI (hemihydrate) Methyl ethylketone Form IEvaporation Screen at 25 and 50° C.

Evaporation studies were carried out to identify the predominant crystalform during uncontrolled precipitation. The compound of Formula I (fromExample 1) was dissolved in a solvent and then the resulting solutionwas subject to evaporation. Specifically, approximately 2 mL ofsaturated solution of the compound of Formula I in various solvents (seeTables 4 and 5 below) were evaporated under air without stirring at25±1° C. and at 50±1° C. controlled by a IKA® ETS-D5 temperaturecontroller and a IKA® RCT basic safety control. Experiments notresulting in any particulate solids were not studied. XRPD was used toidentify the crystalline forms obtained. Results of the screens areindicated below in Tables 4 and 5. The entry “N/A” means that either thesample contained only clear solution or the amount of solid was toosmall to be analyzed by XRPD.

TABLE 4 Evaporation Results at 25° C. Solvent Form Acetonitrile Form IChloroform Oil Methylene chloride Form I Dimethyl formamide N/A1,4-Dioxane Amorphous + solid Methanol Amorphous + solid2-Methoxyethanol N/A Methyl isobutyl ketone Form IX Toluene N/A HexaneN/A Tetrahydrofuran Oil Acetone Form I n-Butyl alcohol N/A Methylt-butyl ether N/A Dimethyl sulfoxide N/A Ethanol Form XIII Ethyl acetateN/A Ethyl formate Amorphous Heptane N/A Isobutyl acetate N/A Isopropylacetate Form III 1-Propanol Form II Isopropyl alcohol Form VI Water N/AMethyl ethyl ketone N/A

TABLE 5 Evaporation Results at 50° C. Solvent Form AcetonitrileAmorphous Chloroform Amorphous Methylene chloride N/A Dimethyl formamideAmorphous 1,4-Dioxane N/A Methanol Amorphous 2-Methoxyethanol Form XIIIMethyl isobutyl ketone N/A Toluene Form IV Hexane N/A TetrahydrofuranAmorphous + solid Acetone Form X n-Butyl alcohol Form VIII Methylt-butyl ether N/A Dimethyl sulfoxide N/A Ethanol Amorphous + solid Ethylacetate N/A Ethyl formate Amorphous Heptane N/A Isobutyl acetate N/AIsopropyl acetate Form III 1-Propanol Amorphous Isopropyl alcohol FormII Water N/A Methyl ethyl ketone AmorphousAntisolvent Addition Screen

Saturated solutions of the compound of Formula I (from Example 1) wereprepared by adding the compound to a solvent at room temperature untilno more solids were dissolved. An antisolvent was added to induceprecipitation. Specifically, the antisolvent was added dropwise at 1-6times volume of solvent. Experiments that did not produce anyparticulate solids were not studied further. The results are presentedin Table 6 below. The entry “N/A” means that either the sample containedonly clear solution or the amount of solid was too small to be analyzedby XRPD.

TABLE 6 Antisolvent Addition Results Solvent (mL) Antisolvent (mL) FormAcetonitrile (1.0) Water (1.5) N/A Chloroform (0.5) Heptane (2.5)amorphous Chloroform (0.5) Hexane (2.5) amorphous Chloroform (0.5)Methyl t-butyl ether (3.0) amorphous Dimethyl formamide Water (1.5) N/A(0.4) Methanol (1.0) Water (3.5) N/A 2-Methoxyethanol Water N/ATetrahydrofuran (1.0) Heptane (3.0) amorphous Tetrahydrofuran (1.0)Hexane (2.5) Form XI Tetrahydrofuran (1.0) Methyl t-butyl ether (3.0)N/A Tetrahydrofuran (1.0) Water (2.5) Form VI Dimethyl sulfoxide WaterN/A Ethanol (0.6) Heptane (3.0) Form II + amorphous Ethanol (0.6) Hexane(3.0) N/A Ethanol (0.5) Methyl t-butyl ether (2.5) N/A Ethanol (0.5)Water (1.0) N/A Isopropyl alcohol (0.5) Heptane (2.5) Form II Isopropylalcohol (0.5) Hexane (2.5) Form II Isopropyl alcohol (0.5) Methylt-butyl ether (3.0) N/A Isopropyl alcohol (0.5) Water (2.0) N/AReverse Addition Screen

Saturated or near saturated solutions (0.5-1 mL) of the compound ofFormula I (from Example I) in various solvents were added to a largervolume of antisolvent. In most cases, no precipitate was obtained.Results are shown in Table 8 below. The entry “N/A” means that eitherthe sample contained only clear solution or the amount of solid was toosmall to be analyzed by XRPD.

TABLE 8 Reverse Addition Results. Solvent Antisolvent Form ChloroformHeptane amorphous Chloroform Hexane amorphous Chloroform Methyl t-butylether amorphous 1,4-Dioxane Heptane N/A 1,4-Dioxane Hexane N/A1,4-Dioxane Methyl t-butyl ether N/A Dimethyl formamide Water Form VIMethanol Water N/A 2-Methoxyethanol Water N/A Tetrahydrofuran HeptaneForm VI Tetrahydrofuran Hexane amorphous Tetrahydrofuran Methyl t-butylether N/A Tetrahydrofuran Water Form VI Dimethyl sulfoxide Water N/AEthanol Heptane N/A Ethanol Hexane N/A Ethanol Methyl t-butyl ether N/AEthanol Water Form VI Isopropyl alcohol Heptane Form II Isopropylalcohol Hexane Form II Isopropyl alcohol Methyl t-butyl ether N/AIsopropyl alcohol Water N/AQuench Cooling Screen

Saturated solutions of the compound of Formula I (from Example I) wereprepared at 30-50° C. and quench cooled to about −15° C. to induceprecipitation. Results of the screen are presented below in Table 9. Theentry “N/A” means that either the sample contained only clear solutionor the amount of solid was too small to be analyzed by XRPD.

TABLE 9 Quench Cooling Results. Solvent Form Acetonitrile N/A Methylisobutyl ketone N/A Toluene Form IV n-Butanol N/A Methyl t-butyl etherN/A Ethanol N/A Ethyl acetate N/A Ethyl formate N/A Isobutyl acetate N/AIsopropyl acetate N/A 1-Propanol N/A Isopropyl alcohol Form IISaturated Solution Heating and Cooling Cycle Screen

Saturated solutions (about 3 mL) of the compound of Formula I (fromExample 1) were prepared at 30 to 50° C. and cooled slowly using aprogrammed circulating bath to form a slurry of solvent and precipitate.This slurry was then heated to 50° C. over 2 hours and then cooled downto 5° C. over 2 hours. The process was repeated overnight and the solidwas isolated for further analysis. The results are presented in Table10. The entry “N/A” means that either the sample contained only clearsolution or the amount of solid was too small to be analyzed by XRPD.

TABLE 10 Heating and Cooling Cycle Results. Solvent Form AcetonitrileN/A Chloroform N/A Methylene chloride Form I Dimethylformamide N/A1,4-Dioxane N/A Methanol N/A 2-Methoxyethanol N/A Methyl isobutyl ketoneForm VII Toluene Form X Hexane Amorphous + solid Tetrahydrofuran N/AAcetone Form X n-Butanol N/A Methyl t-butyl ether N/A DimethylsulfoxideN/A Ethanol N/A Ethyl acetate Form I Ethyl formate N/A Isobutyl acetateForm XI Isopropyl acetate Form II 1-Propanol N/A Isopropyl alcohol FormII Water Form VI Methyl ethyl ketone Form I

Example 3 Experiments Related to Stability of the Crystalline Forms

Competitive Slurry Experiment in Methanol-Ethyl Acetate at ElevatedTemperature

Forms I and X of the compound of Formula I were slurried together in amethanol-ethyl acetate (1:10) solvent system and heated at 50° C. for 5days. Specifically, 5 mL of ethyl acetate and 0.5 nth of methanol werecombined and heated to 50° C. Form I (from Example 1) was added to thesolvent mixture until a cloudy solution formed (about 156 mg), and thenadditional Form I was added (about 50 mg). Then 50 mg of Form X(prepared as described in Example 12) was added. The mixture was stirredat 50° C. for 5 days and the solid was characterized and monitored byXRPD. The resulting crystalline form was predominantly Form I with otherminor forms detected.

Competitive Slurry Experiment of all Forms in Methanol-Acetate at RoomTemperature

Forms I to XIII of the compound of Formula I were slurried together in amethanol-ethyl acetate (1:10) solvent system at room temperature for 8days. Specifically, 10 mg of Form I (from Example 1) was added to 1 mLof ethyl acetate with stirring. Then 0.1 mL of methanol was added givinga cloudy solution to which 3 mg of additional Form I (from Example 1)was added. About 3 mg each of the other crystalline Forms II to XIII,prepared according to the chart below, were then added and the resultingslurry was stirred for 8 days and the solids characterized by XRPD. Theresulting crystalline form detected after 8 days was predominantly FormI.

Methods of Preparation for Forms II to XIII

Form Preparation Form II To 2 mL of saturated solution of Form I in IPAwas added about 30 mg of additional Form I followed by stirring at 50 ±1° C. For 3 days. The solid was centrifuged and characterized by XRPD asForm II. Form III To 2 mL of saturated solution of Form I in IPAc wasadded about 30 mg of additional Form I followed by stirring at 50 ± 1°C. For 3 days. The solid was centrifuged and characterized by XRPD asForm III. Form IV To 2 mL of saturated solution of Form I in toluene wasadded about 30 mg of additional Form I followed by stirring at 25 ± 1°C. For 3 days. The solid was centrifuged and characterized by XRPD asForm IV. Form V To 2 mL of saturated solution of Form I in isobutylacetate was added about 30 mg of additional Form I followed by stirringat 25 ± 1° C. For 3 days. The solid was centrifuged and characterized byXRPD as Form V. Form VI To 2 mL of saturated solution of Form I in waterwas added about 30 mg of additional Form I followed by stirring at 25 ±1° C. For 3 days. The solid was isolated by centrifugation andcharacterized by XRPD as Form VI. Form VII To 2 mL of saturated solutionof Form I in 1,4-dioxane was added about 30 mg of additional Form Ifollowed by stirring at 25 ± 1° C. For 3 days. The solid was centrifugedand characterized by XRPD as Form VII. Form VIII Approximately 2 mL ofsaturated solution of Form I in n-butanol were evaporated under airwithout stirring at 50 ± 1° C. to give solid, which was characterized byXRPD as Form VIII. Form IX To 2 mL of saturated solution of Form I inMIBK was added about 30 mg of additional Form I followed by stirring at50 ± 1° C. For 3 days. The solid was centrifuged and characterized byXRPD as Form IX. Form X To 2 mL of saturated solution of Form I inacetone was added about 30 mg of additional Form I followed by stirringat 50 ± 1° C. For 3 days. The solid was isolated by centrifugation andcharacterized by XRPD as Form X. Form XI Approximately 3 mL of saturatedsolutions Form I in isobutyl acetate was prepared at 30° C. to 50° C.and cooled to 25° C. in a bath slowly by using a programmed circulatingbath. The formed solution was heated to 50° C. over 2 hours and thencooled to 5° C. over 2 hours. This process was repeated for 76 hrs andthe solid was isolated by centrifugation and analyzed by XRPD as FormXI. Form XII To 2 mL of saturated solution of Form I in MTBE was addedabout 30 mg of additional Form I followed by stirring at 50 ± 1° C. For3 days. The solid was centrifuged and characterized by XRPD as Form XII.Form XIII Approximately 2 mL of saturated solution of Form I in2-methoxyethanol were evaporated under air without stirring at 50 ± 1°C. to give solid, which was characterized by XRPD as Form XIII.Competitive Slurry Experiment in Acetone at Elevated Temperature

Forms I and X of the compound of Formula I were slurried together inacetone and heated at 50° C. overnight. Specifically, 5 mL of acetonewas heated to 50° C. Form I (from Example 1) was added to the solventmixture until a cloudy solution formed (about 190 mg), and thenadditional Form I was added (about 50 mg). Then 50 mg of Form X(prepared as described in Example 12) was added. The mixture was stirredat 50° C. overnight and the solid was characterized and monitored byXRPD. The resulting crystalline form was predominantly Form X. Thus,Form I can be converted to Form X under certain conditions.

Competitive Slurry Experiment in Acetone at Room Temperature

Forms I to XIII of the compound of Formula I were slurried together inacetone at room temperature for 11 days. Specifically, 1 mL of acetonewas combined with 11.2 mg of Form I (from Example 1) at room temperatureto give a clear solution, and then additional Form I was added (about 10mg) to give a slurry. Then 0.5 mL of acetone was added to give a cloudysolution. Then about 2 mg each of Forms II-XIII (see above chart) wereadded. The mixture was stirred at room temperature for 11 days and theresulting solid was characterized and monitored by XRPD. The resultingcrystalline form was predominantly Form I with other minor forms alsodetected.

Example 4 Preparation and Characterization of Form II

Form II was prepared as follows. To 0.5 mL of saturated solution of FormI in IPA was added 2.5 mL of heptane followed by stirring to give asolid, which was analyzed by XRPD as Form II.

The XRPD for Form II is provided in FIG. 5 and a list of correspondingpeaks is provided in Table 11 below.

TABLE 11 XRPD Peaks for Form II. 2-Theta Height H % 4.0 277 11.1 7.2 26210.5 9.2 405 16.2 11.1 330 13.2 12.0 106 4.2 14.8 1296 51.7 15.8 500 2016.7 59 2.4 18.5 1927 77 19.3 2504 100 20.3 82 3.3 20.8 604 24.1 21.7533 21.3 22.5 296 11.8 22.8 1243 49.6 23.1 376 15 23.8 142 5.7 24.5 2249 25.0 246 9.8 25.6 780 31.1 26.5 90 3.6 27.8 454 18.1 28.3 273 10.928.7 370 14.8 29.5 66 2.6 30.3 128 5.1 30.7 156 6.2 31.4 122 4.9 32.4144 5.8 34.6 238 9.5 35.9 70 2.8 36.6 154 6.1 37.5 60 2.4

The TGA of Form II is provided in FIG. 6. The sample showed about 0.1%weight loss up to 100° C., and about 11% weight loss between 100 and200° C.

Example 5 Preparation and Characterization of Form III

Form III was prepared as follows. To 2 mL of saturated solution of FormI in IPAc was added about 30 mg of additional Form I followed bystirring at 50±1° C. For 3 days. The solid was centrifuged andcharacterized by XRPD as Form III.

The XRPD spectrum for Form III is provided in FIG. 7 and a list ofcorresponding peaks is provided in Table 12 below

TABLE 12 XRPD Peaks for Form III. 2-Theta Height H % 3.8 284 3.8 10.93624 48.5 11.3 1016 13.6 12.3 1027 13.7 13.9 885 11.8 14.9 495 6.6 15.7716 9.6 17.8 621 8.3 18.6 1087 14.5 19.5 643 8.6 20.1 753 10.1 21.0 118315.8 21.8 7476 100 22.7 1177 15.7 23.1 795 10.6 24.6 1972 26.4 25.1 127217 25.8 65 0.9 26.6 434 5.8 27.2 816 10.9 27.8 53 0.7 28.4 1070 14.328.9 206 2.8 30.3 239 3.2 31.7 436 5.8 32.1 184 2.5 33.0 618 8.3 33.5 751 38.8 239 3.2 39.1 423 5.7

Two DSC thermograms for Form III are provided in FIG. 8. The first is aninitial cycle showing an endotherm with a maximum at about 133° C. Thesecond is a thermogram of the sample after it had already been heated to250° C., and it shows an endotherm with a peak maximum at about 241° C.

The TGA thermogram for Form III is provided in FIG. 9. Form III shows0.2% weight loss up to 100° C., and 19% weight loss between 100 and 200°C.

Example 6 Preparation and Characterization of Form IV

Form IV was prepared as follows. To 2 mL of saturated solution of Form Iin toluene was added about 30 mg of additional Form I followed bystirring at 50±1° C. For 3 days. The solid was centrifuged andcharacterized by XRPD as Form IV.

The XRPD for Form IV is shown in FIG. 10 and a list of correspondingpeaks is provided in Table 13 below.

TABLE 13 XRPD Peaks for Form IV. 2-Theta Height H % 5.9 358 31 8.8 94081.5 9.2 564 48.9 10.5 86 7.5 11.8 133 11.5 12.4 74 6.4 13.4 211 18.314.4 241 20.9 15.3 301 26.1 17.7 1090 94.5 19.5 207 18 23.6 1153 10024.5 140 12.1 25.0 203 17.6 26.4 724 62.8 26.8 867 75.2 27.7 127 11 29.6587 50.9

Two DSC thermograms are shown in FIG. 11 that are representative of FormIV, The first is an initial cycle showing an endothermic event having amaximum at about 153° C. The second thermocycle represents Form IV afterit had been heated to 250° C. An endotherm with a maximum at about 149°C. is shown.

A TGA thermogram representative of Form IV is shown in FIG. 12. Thesample showed 3.4% weight loss up to 100° C., and 8.4% weight lossbetween 100 and 200° C.

Example 7 Preparation and Characterization of Form V

Form V was prepared as follows. To 2 mL of saturated solution of Form Iin isobutyl acetate was added about 30 mg of additional Form I followedby stirring at 25±1° C. For 3 days. The solid was centrifuged andcharacterized by XRPD as Form V.

The XRPD pattern for Form V is shown in FIG. 13 and a list ofcorresponding peaks is provided in Table 14 below.

TABLE 14 XRPD Peaks for Form V. 2-Theta Height H % 3.4 122 3.4 10.6 3319.2 11.1 663 18.5 12.0 1191 33.3 13.6 716 20 14.6 261 7.3 15.4 670 18.715.7 232 6.5 17.5 2351 65.7 18.4 960 26.8 19.3 926 25.9 19.9 1185 33.120.8 1033 28.8 21.3 460 12.8 21.6 347 9.7 22.4 1573 43.9 22.9 3581 10024.2 371 10.4 24.8 1263 35.3 26.4 655 18.3 27.0 729 20.4 28.0 371 10.428.6 893 24.9 29.8 1092 30.5 31.3 183 5.1 33.3 81 2.3 34.3 398 11.1 34.996 2.7 35.6 132 3.7 36.6 305 8.5 37.3 85 2.4 38.4 219 6.1

Two DSC thermograms are shown in FIG. 14 that are representative of FormV. The first thermogram represents an initial DSC cycle that ischaracterized by an endothermic peak maximum at about 121° C. The secondthermogram represents a further DSC cycle carried out after the samplehad already been heated to 250° C., showing an endothermic peak maximumat about 242° C.

A TGA thermogram representative of Form V is shown in FIG. 15. Thesample showed about 0.2% weight loss up to 100° C. and about 21% weightloss between 100 and 200° C.

Example 8 Preparation and Characterization of Form VI

Form VI was prepared by combining about 1.0 g of the compound of FormulaI (from Example 1) with 17 mL of water and then heating the resultingslurry at 50° C. with stirring for 3 days. The predominant crystallineform that was detected was Form VI based on characterization of theresulting solid by XRPD. The XRPD spectrum for Form VI is provided inFIG. 16 and the corresponding peak data is provided below in Table 15.

TABLE 15 XRPD Peak Data for Form VI. 2-Theta Height H % 3.5 173 8.8 3.8172 8.7 10.7 879 44.6 13.2 119 6.1 13.8 205 10.4 14.6 1199 60.8 15.8 42321.5 16.0 762 38.7 16.7 505 25.6 17.8 577 29.3 19.1 1176 59.7 19.9 1457.4 20.8 302 15.3 21.3 187 9.5 22.4 727 36.9 23.9 1971 100 24.5 633 32.125.0 303 15.4 25.5 172 8.7 26.7 902 45.8 27.8 117 5.9 28.3 94 4.8 29.1942 47.8 29.4 203 10.3 30.3 1369 69.5 31.9 118 6 32.5 363 18.4 33.4 1216.2 34.7 448 22.7 35.7 98 5 35.9 129 6.6 36.9 241 12.2 38.0 173 8.8 39.5114 5.8 40.4 170 8.6 41.3 140 7.1

The DSC data for Form VI is provided in FIG. 17, showing the initial DSCthermogram and a second DSC thermogram after the sample had been heatedto 250° C. The initial DSC thermogram showed multiple endotherms withmajor peaks at about 134° C. and about 167° C. The second DSC thermogramshowed a single endotherm with a sharp peak maximum at about 242° C.,indicating that the heating of Form VI can result in a different solidform.

The TGA data for Form VI is provided in FIG. 18 and shows 2.6% weightloss up to about 100° C. and an additional weight loss of 1.3% between100° C. and 250° C. The TGA data indicates that Form VI is likely ahydrated crystalline form of the compound of Formula I, potentially ahemihydrate.

Example 9 Preparation and Characterization of Form VII

Form VII was prepared as follows. To 2 mL of saturated solution of FormI in 1,4-dioxane was added about 30 mg of additional Form I followed bystirring at 25±1° C. For 3 days. The solid was centrifuged andcharacterized by XRPD as Form VII.

The XRPD spectrum for Form VII is provided in FIG. 19 and thecorresponding peak data is provided below in Table 16.

TABLE 16 XRPD Peak Data for Form VII. 2-Theta Height H % 3.6 151 7.3 8.8230 11.1 11.0 252 12.2 12.0 537 26.1 14.4 200 9.7 15.1 761 36.9 15.8 44621.7 16.2 535 26 17.8 2059 100 18.5 817 39.7 19.5 930 45.1 19.9 261 12.721.5 199 9.7 22.1 663 32.2 23.2 108 5.2 24.0 547 26.6 24.6 1655 80.425.3 230 11.2 25.9 1192 57.9 26.7 108 5.3 27.9 298 14.5 29.0 155 7.529.5 155 7.5 30.5 429 20.8 30.8 255 12.4 31.9 307 14.9 32.9 146 7.1 33.7341 16.6 34.1 55 2.7 34.7 63 3.1 39.6 208 10.1 40.5 93 4.5

The TGA thermogram for Form VII is provided in FIG. 20 and shows about0.02% weight loss up to 100° C., and about 11% weight loss between 100and 200° C.

Two DSC thermograms for Form VII are shown in FIG. 21. Cycle 1 shows theinitial thermogram with an endothermic peak having a maximum at about123° C. Cycle 2 shows the thermogram after the sample had been heated to250° C., and shows an endothermic event having a maximum at about 257°C.

Example 10 Preparation and Characterization of Form VIII

Form VIII was prepared as follows. Approximately 2 mL of saturatedsolution of Form I in n-butanol were evaporated under air withoutstirring at 50±1° C. to give solid, which was characterized by XRPD asForm VIII.

The XRPD spectrum for Form VIII is provided in FIG. 22 and thecorresponding peak data is provided below in Table 17.

TABLE 17 XRPD Peak Data for Form VIII. 2-Theta Height H % 3.9 279 15.17.3 158 8.6 8.2 431 23.3 10.9 1034 55.9 11.7 956 51.7 12.5 240 13 14.4658 35.6 15.4 177 9.6 16.0 554 30 16.5 332 18 17.5 1051 56.9 18.2 37320.2 18.5 535 28.9 19.4 270 14.6 19.7 1005 54.4 20.1 274 14.8 21.5 167990.8 22.6 1848 100 23.3 102 5.5 25.0 310 16.8 25.3 619 33.5 26.0 21711.8 26.8 488 26.4 27.7 208 11.2 28.0 382 20.7 28.8 127 6.9 29.4 26914.6 30.0 238 12.9 30.8 353 19.1 31.4 93 5 36.5 62 3.4 37.3 187 10.137.9 83 4.5

Two DSC thermograms for Form VIII are shown in FIG. 23. Cycle 1 showsthe initial thermogram with an endothermic peak having a maximum atabout 176° C. Cycle 2 shows the thermogram after the sample had beenheated to 250° C.

The TGA thermogram for Form VIII is provided in FIG. 24 and shows about0.4% weight loss up to 100° C. and other thermal events.

Example 11 Preparation and Characterization of Form IX

Form IX was prepared as follows. To 2 mL of saturated solution of Form Iin MIBK was added about 30 mg of additional Form I followed by stirringat 50±1° C. For 3 days. The solid was centrifuged and characterized byXRPD as Form IX.

The XRPD spectrum for Form IX is provided in FIG. 25 and thecorresponding peak data is provided below in Table 18.

TABLE 18 XRPD Peak Data for Form IX. 2-Theta Height H % 3.9 264 9.8 11.11022 38 12.2 1363 50.7 13.8 809 30.1 14.9 160 6 15.5 595 22.2 15.9 1595.9 17.4 1802 67.1 17.7 763 28.4 18.6 1129 42 19.3 1516 56.4 19.9 89533.3 20.9 1113 41.4 21.7 823 30.6 22.4 1294 48.2 22.8 2687 100 24.4 97136.1 24.8 2652 98.7 25.6 99 3.7 26.2 191 7.1 26.8 386 14.4 27.3 498 18.528.4 848 31.6 29.0 92 3.4 29.6 581 21.6 30.0 338 12.6 31.3 347 12.9 31.6254 9.5 32.2 85 3.2 34.0 429 16 35.0 231 8.6 35.9 137 5.1 36.4 299 11.137.4 113 4.2 38.2 193 7.2 39.1 164 6.1 43.5 191 7.1

A DSC thermogram for Form IX is shown in FIG. 26. The thermogram ischaracterized by an endothermic event having a maximum at about 258° C.

The TGA thermogram for Form IX is provided in FIG. 27 and shows about0.03% weight loss up to 100° C. and about 18% weight loss between 100and 200° C.

Example 12 Preparation and Characterization of Form X

Form X was prepared by heating a slurry of Form I (from Example 1) inacetone at 50° C. for 2.5 days. Specifically, 1.06 g of the compound ofFormula I was combined at 50° C. with 16 mL of acetone to give a slurry.The temperature of the mixture was maintained at 50° C. for 2.5 days.XRPD confirmed the presence of Form X.

The XRPD spectrum for Form X is provided in FIG. 28 and thecorresponding peak data is provided below in Table 19.

TABLE 19 XRPD Peak Data for Form X. 2-Theta Height H % 3.9 282 12.3 4.2232 10.1 6.8 821 35.8 9.8 80 3.5 10.5 375 16.4 12.5 64 2.8 13.5 844 36.814.8 800 34.9 15.4 332 14.5 17.0 784 34.2 18.0 136 5.9 18.6 351 15.319.3 328 14.3 19.6 776 33.8 20.2 1021 44.5 21.2 154 6.7 21.7 2293 10022.5 76 3.3 23.8 240 10.5 25.0 1076 46.9 25.4 139 6.1 26.3 1149 50.127.1 58 2.5 28.0 155 6.7 28.3 181 7.9 29.6 758 33 30.7 132 5.8 31.1 833.6

The DSC thermogram for Form X is shown in FIG. 29 and is characterizedby an endotherm having a maximum at about 258° C.

The TGA thermograpm for Form X is shown in FIG. 30 and shows about 0.06%weight loss up to 100° C.

Example 13 Preparation and Characterization of Form XI

Form XI was prepared as follows. Approximately 3 mL of saturatedsolutions Form I in isobutyl acetate was prepared at 30° C. to 50° C.and cooled to 25° C. in a bath slowly by using a programmed circulatingbath. The formed solution was heated to 50° C. over 2 hours and thencooled to 5° C. over 2 hours. This process was repeated for 76 hrs andthe solid was isolated by centrifugation and analyzed by XRPD as FormXI.

The XRPD spectrum for Form XI is provided in FIG. 31 and thecorresponding peak data is provided below in Table 20.

TABLE 20 XRPD Peak Data for Form XI. 2-Theta Height H % 3.8 309 8.1 5.8289 7.6 7.0 266 7 7.7 3775 99 11.7 198 5.2 12.4 559 14.7 13.0 112 2.915.5 78 2.1 16.6 693 18.2 17.6 177 4.6 17.9 477 12.5 18.6 359 9.4 19.386 2.3 20.3 2474 64.9 21.3 183 4.8 22.0 155 4.1 22.9 238 6.2 23.4 3813100 24.2 1326 34.8 24.8 270 7.1 26.3 950 24.9 27.1 172 4.5 28.1 81 2.129.2 175 4.6 29.9 713 18.7 31.4 312 8.2 31.9 299 7.8

Two DSC thermograms are shown in FIG. 32, that are representative ofForm XI. The first thermogram is characterized by an endothermic eventhaving a maximum at about 117° C. The second thermogram represents afurther DSC cycle carried out after the sample had already been heatedto 250° C., showing an endothermic peak maximum at about 243° C.

Example 14 Preparation and Characterization of Form XII

Form XII was prepared as follows. To 2 mL of saturated solution of FormI in MTBE was added about 30 mg of additional Form I followed bystirring at 50±1° C. For 3 days. The solid was centrifuged andcharacterized by XRPD as Form XII.

The XRPD spectrum for Form XII is provided in FIG. 33 and thecorresponding peak data is provided below in Table 21.

TABLE 21 XRPD Peak Data for Form XII. 2-Theta Height H % 3.8 83 5.7 6.0147 10.1 7.8 1131 77.3 9.7 131 9 11.9 113 7.7 12.6 392 26.8 14.7 21014.3 15.2 306 21 16.6 559 38.2 18.2 865 59.1 18.8 185 12.6 19.3 85 5.820.1 889 60.8 22.1 83 5.7 23.0 146 10 23.7 1463 100 25.0 140 9.6 25.9187 12.8 27.3 120 8.2 29.5 354 24.2 29.8 273 18.7 30.7 109 7.4 31.4 936.4 34.3 56 3.8 34.8 113 7.7

A DSC thermogram for Form XII is shown in FIG. 34. The thermogram ischaracterized by an endothermic event having a maximum at about 137° C.

Example 15 Preparation and Characterization of Form XIII

Form XIII was prepared as follows. Approximately 2 mL of saturatedsolution of Form I in 2-methoxyethanol were evaporated under air withoutstirring at 50±1° C. to give solid, which was characterized by XRPD asForm XIII.

The XRPD spectrum for Form XIII is provided in FIG. 35 and thecorresponding peak data is provided below in Table 22.

TABLE 22 XRPD Peak Data for Form XIII. 2-Theta Height H % 3.8 302 30.95.9 845 86.5 7.7 112 11.5 10.1 977 100 11.3 231 23.6 11.7 238 24.3 13.0717 73.3 14.2 103 10.5 14.8 153 15.7 16.2 375 38.3 16.9 564 57.7 17.8237 24.3 18.5 116 11.9 19.4 188 19.2 20.4 153 15.7 21.0 207 21.1 21.6 646.6 22.6 559 57.2 23.4 747 76.4 24.2 229 23.5 24.6 175 17.9 25.4 17718.1 26.0 514 52.6 26.9 704 72.1 28.2 291 29.8 29.5 273 28 30.5 202 20.7

A DSC thermogram for Form XIII is shown in FIG. 36. The thermogram ischaracterized by an endothermic event having a maximum at about 156° C.and a second endotherm at about 238° C.

Example A1 PI3K Enzyme Assay

PI3-Kinase luminescent assay kit including lipid kinase substrate,D-myo-phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)D(+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked (PIP2),biotinylated I(1,3,4,5)P4, PI(3,4,5)P3 Detector Protein, is purchasedfrom Echelon Biosciences (Salt Lake City, Utah). AlphaScreen™ GSTDetection Kit including donor and acceptor beads is purchased fromPerkinElmer Life Sciences (Waltham, Mass.). PI3Kδ (p110δ/p85α) ispurchased from Millipore (Bedford, Mass.). ATP, MgCl₂, DTT, EDTA, HEPESand CHAPS are purchased from Sigma-Aldrich (St. Louis, Mo.).

AlphaScreen™ Assay for PI3Kδ

The kinase reaction is conducted in 384-well REMP plate from ThermoFisher Scientific in a final volume of 40 μL. Inhibitors are firstdiluted serially in DMSO and added to the plate wells before theaddition of other reaction components. The final concentration of DMSOin the assay is 2%. The PI3K assays are carried out at room temperaturein 50 mM HEPES, pH 7.4, 5 mM MgCl₂, 50 mM NaCl, 5 mM DTT and CHAPS0.04%. Reactions are initiated by the addition of ATP, the finalreaction mixture consists of 20 μM PIP2, 20 μM ATP, 1.2 nM PI3Kδ and areincubated for 20 min. 10 μL of reaction mixture is then transferred to 5μL 50 nM biotinylated I(1,3,4,5)P4 in quench buffer: 50 mM HEPES pH 7.4,150 mM NaCl, 10 mM EDTA, 5 mM DTT, 0.1% Tween-20, followed with theaddition of 10 μL AlphaScreen™ donor and acceptor beads suspended inquench buffer containing 25 nM PI(3,4,5)P3 detector protein. The finalconcentration of both donor and acceptor beads is 20 mg/mL. After platesealing, the plate is incubated in a dark location at room temperaturefor 2 hours. The activity of the product is determined on Fusion-alphamicroplate reader (Perkin-Elmer). IC₅₀ determination is performed byfitting the curve of percent control activity versus the log of theinhibitor concentration using the GraphPad Prism 3.0 software.

Example A2 PI3K Enzyme Assay

Materials: Lipid kinase substrate, phosphoinositol-4,5-bisphosphate(PIP2), is purchased from Echelon Biosciences (Salt Lake City, Utah).PI3K isoforms α, β, δ and γ are purchased from Millipore (Bedford,Mass.). ATP, MgCl₂, DTT, EDTA, MOPS and CHAPS are purchased fromSigma-Aldrich (St. Louis, Mo.).

The kinase reaction is conducted in clear-bottom 96-well plate fromThermo Fisher Scientific in a final volume of 24 μL. Inhibitors arefirst diluted serially in DMSO and added to the plate wells before theaddition of other reaction components. The final concentration of DMSOin the assay is 0.5%. The PI3K assays are carried out at roomtemperature in 20 mM MOPS, pH 6.7, 10 mM MgCl₂, 5 mM DTT and CHAPS0.03%. The reaction mixture is prepared containing 50 μM PIP2, kinaseand varying concentration of inhibitors. Reactions are initiated by theaddition of ATP containing 2.2 μCi [γ-³³P]ATP to a final concentrationof 1000 μM. The final concentration of PI3K isoforms α, β, δ and γ inthe assay are 1.3, 9.4, 2.9 and 10.8 nM respectively. Reactions areincubated for 180 min and terminated by the addition of 100 μL of 1 Mpotassium phosphate pH 8.0, 30 mM EDTA quench buffer. A 100 μL aliquotof the reaction solution is then transferred to 96-well MilliporeMultiScreen IP 0.45 μm PVDF filter plate (The filter plate is pre-wettedwith 200 μL 100% ethanol, distilled water, and 1 M potassium phosphatepH 8.0, respectively). The filter plate is aspirated on a MilliporeManifold under vacuum and washed with 18×200 μL wash buffer containing 1M potassium phosphate pH 8.0 and 1 mM ATP. After drying by aspirationand blotting, the plate is air dried in an incubator at 37° C.overnight. Packard TopCount adapter (Millipore) is then attached to theplate followed with addition of 120 μL Microscint 20 scintillationcocktail (Perkin Elmer) in each well. After the plate sealing, theradioactivity of the product is determined by scintillation counting onTopcount (Perkin-Elmer). IC₅₀ determination is performed by fitting thecurve of percent control activity versus the log of the inhibitorconcentration using the GraphPad Prism 3.0 software. Compounds havingand IC₅₀ value of 10 μM or less are considered active.

Example A3 PI3Kδ Scintillation Proximity Assay

Materials

[γ-³³P]ATP (10 mCi/mL) is purchased from Perkin-Elmer (Waltham, Mass.).Lipid kinase substrate, D-myo-Phosphatidylinositol 4,5-bisphosphate(PtdIns(4,5)P2)D (+)-sn-1,2-di-O-octanoylglyceryl, 3-O-phospho linked(PIP2), CAS 204858-53-7, is purchased from Echelon Biosciences (SaltLake City, Utah). PI3Kδ (p110δ/p85α) is purchased from Millipore(Bedford, Mass.). ATP, MgCl₂, DTT, EDTA, MOPS and CHAPS are purchasedfrom Sigma-Aldrich (St. Louis, Mo.). Wheat Germ Agglutinin (WGA) YSi SPAScintillation Beads is purchased from GE healthcare life sciences(Piscataway, N.J.).

The kinase reaction is conducted in polystyrene 384-well matrix whiteplate from Thermo Fisher Scientific in a final volume of 25 μL.Inhibitors are first diluted serially in DMSO and added to the platewells before the addition of other reaction components. The finalconcentration of DMSO in the assay is 0.5%. The PI3K assays are carriedout at room temperature in 20 mM MOPS, pH 6.7, 10 mM MgCl₂, 5 mM DTT andCHAPS 0.03%. Reactions are initiated by the addition of ATP, the finalreaction mixture consisted of 20 μM PIP2, 20 μM ATP, 0.2 μCi [γ-³³P]ATP, 4 nM PI3Kδ. Reactions are incubated for 210 min and terminated bythe addition of 40 μL SPA beads suspended in quench buffer: 150 mMpotassium phosphate pH 8.0, 20% glycerol. 25 mM EDTA, 400 μM ATP. Thefinal concentration of SPA beads is 1.0 mg/mL. After the plate sealing,plates are shaken overnight at room temperature and centrifuged at 1800rpm for 10 minutes, the radioactivity of the product is determined byscintillation counting on Topcount (Perkin-Elmer). IC₅₀ determination isperformed by fitting the curve of percent control activity versus thelog of the inhibitor concentration using the GraphPad Prism 3.0software.

Example B1 B Cell Proliferation Assay

To acquire B cells, human PBMC are isolated from the peripheral blood ofnormal, drug free donors by standard density gradient centrifugation onFicoll-Hypague (GE Healthcare, Piscataway, N.J.) and incubated withanti-CD19 microbeads (Miltenyi Biotech, Auburn, Calif.). The B cells arethen purified by positive immunosorting using an autoMacs (MiltenyiBiotech) according to the manufacturer's instruction.

The purified B cells (2×10⁵/well/200 μL) are cultured in 96-wellultra-low binding plates (Corning, Corning, N.Y.) in RPMI1640, 10% FBSand goat F(ab′)2 anti-human IgM (10 μg/ml) (Invitrogen, Carlsbad,Calif.), in the presence of different amount of test compounds, forthree days. [³H]-thymidine (1 μci/well) (PerkinElmer, Boston, Mass.) inPBS is then added to the B cell cultures for an additional 12 hrs beforethe incorporated radioactivity is separated by filtration with waterthrough GF/B filters (Packard Bioscience, Meriden, Conn.) and measuredby liquid scintillation counting with a TopCount (Packard Bioscience).Compounds having an IC₅₀ value of 10 μM or less are considered active.

Example B2 Pfeiffer Cell Proliferation Assay

Pfeiffer cell line (diffuse large B cell lymphoma) is purchased fromATCC (Manassas, Va.) and maintained in the culture medium recommended(RPMI and 10% FBS). To measure the anti-proliferation activity of thePI3Kδ submittals, the Pfeiffer cells are plated with the culture medium(2×10³ cells/well/per 200 μl) into 96-well ultra-low binding plates(Corning, Corning, N.Y.), in the presence or absence of a concentrationrange of test compounds. After 3-4 days, [³H]-thymidine (1 μCi/well)(PerkinElmer, Boston, Mass.) in PBS is then added to the cell culturefor an additional 12 hrs before the incorporated radioactivity isseparated by filtration with water through GF/B filters (PackardBioscience, Meriden, Conn.) and measured by liquid scintillationcounting with a TopCount (Packard Bioscience).

Example C Akt Phosphorylation Assay

Ramos cells (B lymphocyte from Burkitts lymphoma) can be obtained fromATCC (Manassas, Va.) and maintained in RPMI1640 and 10% FBS. The cells(3×10⁷ cells/tube/3 mL in RPMI) are incubated with different amounts oftest compounds for 2 hrs at 37° C. and then stimulated with goat F(ab′)2anti-human IgM (5 μg/mL) (Invitrogen) for 17 min. in a 37° C. waterbath. The stimulated cells are spun down at 4° C. with centrifugationand whole cell extracts prepared using 300 μL lysis buffer (CellSignaling Technology, Danvers, Mass.). The resulting lysates aresonicated and supernatants are collected. The phosphorylation level ofAkt in the supernatants are analyzed by using PathScan phospho-Akt1(Ser473) sandwich ELISA kits (Cell Signaling Technology) according tothe manufacturer's instruction.

Example D In vitro JAK Kinase Assay

The compounds in Table A were tested for inhibitory activity of JAKtargets according to the following in vitro assay described in Park etal., Analytical Biochemistry 1999, 269, 94-104. The catalytic domains ofhuman JAK1 (a.a. 837-1142), JAK2 (a.a. 828-1132) and JAK3 (a.a.781-1124) were expressed using baculovirus in insect cells and purified.The catalytic activity of JAK1, JAK2 or JAK3 was assayed by measuringthe phosphorylation of a biotinylated peptide. The phosphorylatedpeptide was detected by homogenous time resolved fluorescence (HTRF).IC₅₀s of compounds were measured for each kinase in the 40 μL reactionsthat contain the enzyme, ATP and 500 nM peptide in 50 mM Tris (pH 7.8)buffer with 100 mM NaCl, 5 mM DTT, and 0.1 mg/mL (0.01%) BSA. For the 1mM IC₅₀ measurements, ATP concentration in the reactions was 1 mM.Reactions were carried out at room temperature for 1 hour and thenstopped with 20 μL 45 mM EDTA, 300 nM SA-APC, 6 nM Eu-Py20 in assaybuffer (Perkin Elmer, Boston, Mass.). Binding to the Europium labeledantibody took place for 40 minutes and HTRF signal was measured on aPHERA star plate reader (BMG, Cary, N.C.). The data for the JAK1 and/orJAK2 inhibitors were obtained by testing the compounds in the Example Dassay at 1 mM ATP.

What is claimed is:
 1. A crystalline form of the compound(S)-7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-onewhich is hydrated.
 2. The crystalline form of claim 1 which is ahemihydrate.
 3. The crystalline form of claim 1 which is Form VI.
 4. Thecrystalline form of claim 1 having an X-ray powder diffraction patterncomprising at least one peak, in terms of 2θ, at 10.7°±0.2°.
 5. Thecrystalline form of claim 1 having an X-ray powder diffraction patterncomprising the following peaks, in terms of 2θ: 10.7°±0.2°; 14.6°±0.2°;19.1°±0.2°; and 23.9°±0.2°.
 6. The crystalline form of claim 1 having anX-ray powder diffraction pattern comprising 4 or more of the followingpeaks, in terms of 2θ: 10.7°±0.2°; 14.6°±0.2°; 16.0°±0.2°; 19.1°±0.2°;22.4 ±0.2″; 23.9°±0.2°; 24.5 ±0.2°; 26.7°±0.2°; 29.1°±0.2°; 30.3°±0.2°;and 34.7°±0.2°.
 7. The crystalline form of claim 1 having an X-raypowder diffraction pattern substantially as shown in FIG.
 16. 8. Thecrystalline form of claim 1 having a differential scanning calorimetrythermogram (DSC) substantially as shown in FIG. 17 (upper, Cycle 1). 9.The crystalline form of claim 1 having a thermogravimetric analysis(TGA) substantially as shown in FIG.
 18. 10. The crystalline form ofclaim 1 which is substantially isolated.
 11. A composition comprisingthe crystalline form of claim
 1. 12. The composition of claim 11 whereinsaid composition comprises at least one pharmaceutically acceptablecarrier.
 13. A process for preparing the crystalline form of claim 1comprising combining the compound(S)-7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-onewith water.
 14. The process of claim 13 further comprising heating themixture resulting from the combining of said compound and water.
 15. Acrystalline form of the compound(S)-7-(1-(9H-purin-6-ylamino)ethyl)-6-(3-fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-onewhich is Form X.
 16. The crystalline form of claim 15 having an X-raypowder diffraction pattern comprising at least one peak, in terms of 2θ,at 6.8°±0.2°.
 17. The crystalline form of claim 15 having an X-raypowder diffraction pattern comprising the following peaks, in terms of2θ: 6.8°±0.2°; 13.5°±0.2°; 20.2°±0.2°; and 21.7°±0.2°.
 18. Thecrystalline form of claim 15 having an X-ray powder diffraction patterncomprising 4 or more of the following peaks, in terms of 2θ: 6.8°±0.2°;10.5°±0.2°; 13.5°±0.2°; 14.8°±0.2°; 17.0°±0.2°; 19.6°±0.2°; 20.2°±0.2°;21.7°±0.2′; 25.0°±0.2′; 26.3°±0.2°; and 29.6°±0.2°.
 19. The crystallineform of claim 15 having an X-ray powder diffraction patternsubstantially as shown in FIG.
 28. 20. The crystalline form of claim 15having a differential scanning calorimetry (DSC) thermogramcharacterized by an endotherm at 258±4° C.
 21. The crystalline form ofclaim 15 having a differential scanning calorimetry thermogram (DSC)substantially as shown in FIG.
 29. 22. The crystalline form of claim 15having a thermogravimetric analysis (TGA) substantially as shown in FIG.30.
 23. The crystalline form of claim 15 which is substantiallyisolated.
 24. A composition comprising the crystalline form of claim 15.25. The composition of claim 24 wherein said composition comprises atleast one pharmaceutically acceptable carrier.
 26. A process forpreparing the crystalline form of claim 15 comprising combiningcrystalline Form I of the compound(S)-7-(1-(9H-purin-6-ylamino)ethyl)-6-(fluorophenyl)-3-methyl-5H-thiazolo[3,2-a]pyrimidin-5-one with acetone.
 27. The process of claim 26 furthercomprising heating the mixture resulting from the combining of saidcrystalline Form I and acetone.